Methods and devices related to amplifying nucleic acid at a variety of temperatures

ABSTRACT

Disclosed are compositions and methods for nucleic acid amplification and detection. Specifically, disclosed herein are compositions and methods that allow for amplification of nucleic acids at a wide variety of temperatures. This includes a polymerase which is thermostable at high temperatures, and a method of amplification that can be conducted at relatively low temperatures.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 16/612,020, filed Nov. 8, 2019, which is a NationalPhase Application of PCT/US2018/032074, filed May 10, 2018, and claimsbenefit of U.S. Provisional Application No. 62/504,250, filed May 10,2017, which are hereby incorporated by reference in their entirety.

ACKNOWLEDGEMENTS

This invention was made with government support under Grant No. GM128446awarded by the National Institutes of Health, Grant Nos. CMMI1761743 and1541244 awarded by the National Science Foundation, Grant No.W911NF-17-1-0561 awarded by the Army Research Office, Grant No.80NSSC17K0520 awarded by National Aeronautics and Space Administration(NASA), and Grant No. FA9550-14-1-0089 awarded by the Air Force Officeof Scientific Research. The government has certain rights in theinvention.

BACKGROUND A. Background for Thermophilic Strand-Displacing Polymerase

Over the last several decades, isothermal nucleic acid amplification(IA) has become a transformative technology for point of carediagnostics that seek to deliver clinical results to patients in nearreal-time (Craw 2012; Hartman 2013). Because IA methods seek to amplifyDNA or RNA via continuous replication at a single temperature, theyobviate the need for thermal cyclers and can reduce the time to result(Craw 2012; Gill 2008; Mori 2013; Njiru 2012; Zhao 2015). These assayadvantages have in turn enabled the creation of a variety of fascinatingand useful point of care devices (Craw 2012; Hartman 2013; Mori 2013;Zhao 2015; Asiello 2011; Du 2015; Jiang 2015).

While some IA mechanisms depend upon multiple enzymes, includingnickases, recombinases, and ligases, to achieve continuous replication,rolling circle amplification (RCA) and loop-mediated isothermalamplification (LAMP) require only polymerases and primers (Gill 2008;Notomi 2000; Dean 2001). RCA can proceed at mesophilic or highertemperatures, amplifying continuously around a circular template togenerate long, concatenated DNA products (Jiang 2013). When initiatedfrom a nick or single primer, amplification is linear; by including bothforward and reverse primers, however, amplification becomes exponential,generating 10⁹-fold amplification in 90 minutes from 10 copies oftemplate in a reaction commonly referred to as hyperbranched RCA (hbRCA)(Craw 2012; Dean 2001; Lizardi 1998; Zhang 2001). LAMP, alsoexponential, is currently an inherently higher temperature mechanism,using 4-6 primers to generate 10⁹-fold amplification of short (100-500bp) DNA targets in an hour or less by creating ladder-like concatenatedamplicons (Njiru 2012; Notomi 2000; Bhadra 2015). Overall, both methodsare rapid, single-enzyme DNA detection systems that are comparable toPCR in terms of sensitivity, yet are faster and can operateisothermally, likely explaining their prevalence in point of care assaysand devices (Craw 2012; Zhao 2015).

Like many IA strategies, LAMP and RCA rely upon the inherent stranddisplacement activity of a polymerase to displace downstream DNA,thereby enabling continuous replication without thermal cycling (Gill2008; Zhao 2015). There are only a limited number of polymerases withstrong strand displacement characteristics, primarily the large fragment(exo-) of Geobacillus stearothermophilus pol I (Bst LF) for hightemperature reactions (65-70° C., (Kiefer 1997)) or the Bacillussubtilis phage phi29 polymerase (429) for low temperature reactions(≤30° C., (Blanco 1989)). These polymerases are also highly processive(Kiefer 1997; Blanco 1989), a property that often coincides with stranddisplacement and that makes them useful for sequencing otherwisedifficult DNA molecules (McClary 1991; Ye 1987; Zhang 2006).

Unfortunately, while many IA mechanisms depend upon an initial heatingstep (˜95° C.) for template denaturation (Craw 2012), both 29 and Bst LFare denatured at much lower temperatures (phi29 at 37° C., Bst at 80°C.). Thus, some IA reactions require opening reaction tubes and addingpolymerase after the heating step, which is both cumbersome and riskydue to the common issues of spurious amplification andcross-contamination inherent in ultrasensitive IA strategies (Craw 2012;Hsieh 2014). While many IA mechanisms including LAMP and some versionsof RCA do not necessarily require template denaturation, pre-reactionheating can nonetheless improve assay sensitivity (Njiru 2008; Suzuki2010), reduce amplification inhibition from crude clinical samples(Verkooyen 1996; Modak 2016), and serve as a nucleic acid extractionmethod for detection of viruses and bacteria (Fereidouni 2015;Queipo-Ortuno 2008). Thus, there is a pressing need for thermostablepolymerases that possess significant strand displacement activity forboth IA and PCR methods.

B. Background for Phosphorothioated Loop-Mediated IsothermalAmplification (PS-LAMP)

Loop-mediated isothermal amplification (LAMP) introduced by Notomi etal. (Notomi 2000) is a highly sensitive, specific and rapid method forDNA amplification at a constant temperature (60-65° C.) (Nagamine, 2002;Tomita 2008; Nagamine 2002; Mair 2013; Tanner 2012) LAMP can achieve10⁹-fold amplification within one hour, relying on auto-cycling stranddisplacement DNA synthesis. It is performed with a strand-displacingBacillus stearothermophilus (Bst) DNA polymerase and four to sixspecially designed primers that enable the highly specific recognitionof target DNA, generating amplicons that have a loop structure that canbe utilized for sequence-specific signaling (Jiang 2015). LAMP has alsoproven useful for the detection of many infectious agents includingdisease-causing parasites and other pathogens (Suwancharoen 2016; Wang2016; Abdulmawjood 2016; Song 2016; Kong 2016; Zhang 2016).

Although LAMP is a valuable tool for the detection of nucleic acids,LAMP's applicability would be remarkably improved if it could beperformed at low temperatures. Working at low temperatures could improvedetection of target variants by enabling mismatched (degenerate) primersto bind better. Additionally, it could reduce device complexity andpower consumption when incorporated into portable devices that areuseful for point of care applications. Reduced heating needs would meanless power consumption and hence less cost. Several isothermalamplification methods at low temperatures (around 40° C.) such asnucleic acid sequence-based amplification (NASBA), strand displacementamplification (SDA), rolling circle amplification (RCA),helicase-dependent amplification (HDA) and recombinase polymeraseamplification (RPA) have been reported. However, those methods requireadditional steps or enzymes and show relatively low specificity withonly two primers, while LAMP is performed by only one enzyme with a highspecificity derived from four to six primers. What is needed in the artis a version of LAMP which works at lower temperatures.

SUMMARY

Disclosed herein is a non-naturally occurring thermostable polymerase,wherein the thermostable polymerase is characterized by increasedtemperature stability in the range of 70° C. to 100° C., increasedstrand displacement capability, increased processivity, or a combinationthereof compared with a wild type large fragment Bacillusstearothermophilus (Bst LF) polymerase.

Also disclosed herein is a method of identifying a non-naturallyoccurring thermostable polymerases, the method comprising: a) providinga pool of nucleic acids comprising nucleic acid members each encodingnon-naturally occurring, potential thermostable polymerases; b)subdividing the pool of nucleic acid members into cellular compartmentsby transformation into a bacterial host, such that each cell comprises anucleic acid member; c) expressing the nucleic acid member in the cellcompartment to form a potential thermostable polymerase encoded by thenucleic acid member; d) subdividing the pool of bacterial cells intocompartments, such that each compartment contains a single cell with asingle nucleic acid member and encoded polymerase e) subjecting the poolof nucleic acid members to thermal denaturation and isothermalamplification conditions, such that the nucleic acid member may beprocessed by the a thermostable polymerase encoded by said nucleic acidmember; and f) detecting processing of the nucleic acid member by athermostable polymerase encoded by said nucleic acid member, therebyidentifying a thermostable polymerase.

Further disclosed herein is a method of amplifying a nucleic acid, themethod comprising exposing a target nucleic acid to a buffer solutionwith a polymerase and at least four primers, wherein at least one of thefour primers comprises a phosphorothioated nucleotide; and amplifyingthe target nucleic acid using an isothermal amplification reaction,wherein the isothermal amplification reaction produces at least one loopproduct, wherein at least part of the single-stranded portion of theloop product represents the target nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments and togetherwith the description illustrate the disclosed compositions and methods.

FIG. 1 shows an Isothermal CSR schematic. In IsoCSR, E. coli cellsexpressing a plasmid-encoded polymerase library are suspended in awater-in-oil emulsion with a single cell per compartment, preservinggenotype-phenotype linkage. Each compartment contains either lysozymeand Nb.BsmI nickase for enzymatic lysis and RCA initiation inmesothermophilic IsoCSR (A) or primers for heat-initiated lysis and RCAin thermostable IsoCSR (B). Following lysis, functional polymerasesreplicate their own plasmid via isothermal Rolling Circle Amplification,which is dependent upon a polymerase having strong strand-displacingactivity. The most active polymerases (green) produce more DNA, whileless active variants (yellow) produce less; non-functional variants(red) produce none. After the hbRCA reaction, emulsions are broken, DNAis pooled, and the library is recovered by PCR, enriched for functionalvariants by the positive feedback loop in emulsio. The library can befurther recloned into the expression vector for subsequent rounds asneeded.

FIG. 2 shows mock IsoCSR selection with wild-type Bst LF. The IsoCSRselection was initially optimized using wild-type Bst LF and BstXX, aninactive variant with 6 stop codons and a unique EcoRI restriction site.BL21 cells carrying plasmids encoding either polymerase were culturedwith increasing ratios of inactive BstXX:Bst LF, then subjected tomesothermophilic IsoCSR to accommodate BstLF (see FIG. 1a ). Following 2rounds of recovery PCR (20 cycles each), products were digested withEcoRI to distinguish Bst LF from BstXX and analyzed with gelelectrophoresis.

FIG. 3 shows isothermal screening of evolved variants. LAMP activity oftwo functional variants isolated from IsoCSR compared to Bst LF (A).LAMP activity after heating fully assembled LAMP reactions for 1 min ata range of temperatures (B). Solid and dashed lines indicate thetemperature at which the reaction was heated prior to LAMP.

FIG. 4 shows thermostability kinetics of v5.9. Thermostability ofVariant 5.9 was characterized by examining the polymerase's reactionrate in an extension assay after pre-heating at various temperatures andtimes. Activity is normalized to enzyme activity without heating. Dashesindicate temperature. A light grey line indicates ½ life (50% activity).

FIG. 5 shows Rolling Circle Amplification (RCA) with V5.9, Bst LF, andKlentaq. Enzymes were incubated with nicked template (A, linearamplification), nicked template with forward and reverse primers (B,exponential amplification), or supercoiled plasmid with forward andreverse primers to mimic the RCA reaction in our selections (C,exponential amplification). Polymerase colors are coordinated betweengraphs and with other figures. Dashes indicate negative controls.

FIG. 6 shows structural characteristics of chimeric variant 5.9. Thecrystal structure alignment of wild-type Bst LF and Klentaq is pictured(A). The polymerase domain consists of 3 subdomains, termed the fingers,palm, and thumb. The I and H helix of the thumb subdomain form anessential coiled-coil structure. Variant 5.9 consists of the Klentaqprotein sequence with 13 mutations (B), largely comprising a Bstinsertion located at the base of the thumb subdomain. This region ishighly divergent between Klentaq and Bst LF. In Klentaq (C), the regionis largely unstructured; the site of the Bst insertion (blue) containsonly a small alpha helix (*) that is separated from the I helix by anunstructured loop. The nearby E322G mutation we observed in variant 5.9is also pictured (red). In Bst (D), the inserted region (blue) is anextension of the I helix, and contains a beta strand that is part of asmall, antiparallel beta sheet (three black arrows) not present in theKlentaq structure.

FIG. 7 shows program architecture of shuffle optimizer. Thefunctionality of Shuffle Optimizer, a program used to generate optimizedDNA sequences for library shuffling, is summarized. The program isavailable as open-source python code.

FIG. 8 shows results from DNA shuffling of Klentaq and Bst LF. 20individual variants were sequenced from the shuffled library. Thelocation and number of crossovers observed is summarized (A). Eachindividual variant is also pictured (B).

FIG. 9 shows exonuclease III treatment and Taq recovery of contaminatedproducts. An equimolar mixture of 3 dsDNA molecules of sizes 647 bp,1,228 bp, and 1,717 bp was subjected to ExoIII digestion at 25° C. forvarious times, then mixed with Taq and incubated at 68° C. for 10 min.The exonuclease breaks down the bands synchronously in the 3′ to 5′direction, while the subsequent Taq extension recovers bands that stillhave overhangs. Increasingly large bands are eliminated as digest timeis increased. While the smallest band appears to re-emerge in laterreactions, this is actually the breakdown product from the digestedmiddle band. This treatment is effective for removing parasites from PCRreactions.

FIG. 10 shows the melt curve analysis of qLAMP screening. LAMP reactionswere subjected to a melt curve analysis on the LightCycler 96 qPCRmachine (Roche) following isothermal LAMP incubation for productspecificity analysis (see FIG. 4 for data). All products have similarmelt peaks, including those from heat-treated v5.9 reactions.

FIGS. 11A and 11B show LAMP amplification with Bst LF and SD Pol. SD Pol(Bioron), a commercially available Taq mutant reportedly capable of LAMP(Ignatov 2014) was compared with other variants in an initial qLAMPscreening (11A). Template is included as indicated. Using manufacturerrecommended conditions, Amplicons were not generated with SD Pol. Othertemplates were also attempted. SD pol is capable of Rolling CircleAmplification from a nicked plasmid template (11B).

FIG. 12 shows LAMP activity comparison of v5.9 after 1 or 2 min ofheating. Variant 5.9 activity was assayed using qLAMP reactionsidentical to those in the main text with 1 or 2 minutes of pre-reactionheating at the indicated temperatures. Results are similar at thesetemperatures whether heated for 1 or 2 min, with 89.5° C. producing morerapid amplification. This can be due to improved template denaturation.

FIGS. 13 A-C show a melt curve analysis of RCA Reactions. RCA reactionswere subjected to a melt curve analysis on the LightCycler 96 qPCRmachine (Roche) following isothermal incubation for product specificityanalysis. Letters correspond to data in FIG. 5. Linear RCA producessmall melt curves that are similar between all products and correlatewith amount of product (A). Results are similar for hyperbranched RCAfrom the relaxed, nicked plasmid template (B). The v5.9 no templatecontrol shows a different melt peak, indicating non-specificamplification, likely due to primer dimers. Variant 5.9 in the onlypolymerase able to produce exponential amplification from supercoiledplasmid template; this is indicated by the quantity and melt temperatureof product (C). Bst LF produces a melt peak with reduced Tm, overlappingwith a small peak in v5.9.

FIGS. 14 A-C show an in-emulsion cell lysis of E. coli cells expressingGFP using lysozyme. E. coli cells expressing GFP polymerase wereemulsified using standard IsoCSR emulsion conditions (see Methods).Cells were incubated with or without Lysozyme for the times indicated,then imaged using an inverted fluorescent microscope. Cells show verylittle lysis at 4° C. even with lysozyme included for up to 1 hourincubations, as indicated by the tightly confined GFP signal (FIGS. 14Aand 14B, respectively). After digestion with lysozyme for 30 min at 37°C. followed by 30 min at 65° C. (simulating isothermal incubation),cells have been completely lysed, as indicated by GFP occupying theentirety of each emulsion compartment (FIG. 14C).

FIG. 15 shows emulsion size stability after incubation at 65° C.Emulsions were assembled as previously indicated with either 1× or 2×aqueous component volume, emulsified using a TissueLyser (Qiagen) withthe indicated Hz settings for 4 min, and subjected to incubations at 65°C. in order to simulate isothermal amplification conditions. Compartmentsizes were calculated as pixel area using MATLAB software (MathWorks).While the smaller emulsions generated by 42 Hz emulsification appearedstable for up to 6 hr, emulsifications at either frequency were stablefor 3 hrs. 35 Hz emulsions had a larger average and maximum size after 3hr incubations, indicating that these larger compartments are stable.

FIG. 16 shows a scheme of a phosphorothioated loop-mediated isothermalamplification (PS-LAMP).

FIGS. 17 A-C show a comparison of R-LAMP and PS-LAMP with differenttemperatures. (a) Under the same buffer condition, R-LAMP and PS-LAMPwere performed with different temperatures (line 1: notemplate/R-LAMP/65° C., line 2: 1.2×10⁸ copies of template/R-LAMP/65°C., line 3: no template/PS-LAMP/65° C., line 4: 1.2×10⁸ copies oftemplate/PS-LAMP/65° C., line 5: no template/R-LAMP/60° C., line 6:1.2×10⁸ copies of template/R-LAMP/60° C., line 7: notemplate/PS-LAMP/60° C., line 8: 1.2×10⁸ copies of template/PS-LAMP/60°C.). (b) Fluorescence intensities at a 50 cycle for R-LAMP and PS-LAMPwith a 500 pg of template were compared according to differenttemperatures. (c) At 45° C., R-LAMP with a normal buffer and PS-LAMPwith a semi-optimized buffer (12 U of Bst 2.0 DNA polymerase, 0.5 μg ofET SSB and 2 mM of MgSO₄) were performed.

FIGS. 18 A-D show the effects of urea and Bst 2.0 DNA polymerase.Fluorescence intensities were monitored with different concentrations ofurea (line 1: 0 M/no template, line 2: 0 M/1.2×10⁸ copies of template,line 3: 0.48 M/no template, line 4: 0.48 M/1.2×10⁸ copies of template,line 5: 0.96 M/no template, line 6: 0.96 M/1.2×10⁸ copies of template,line 7: 1.2 M/no template, line 8: 1.2 M/1.2×10⁸ copies of template,line 9: 1.44 M/no template, line 10: 1.44 M/1.2×10⁸ copies of template)(a) and units of Bst 2.0 DNA polymerase (line 1: 20 U/no template, line2: 20 U/1.2×10⁸ copies of template, line 3: 40 U/no template, line 4: 40U/1.2×10⁸ copies of template, line 5: 60 U/no template, line 6: 60U/1.2×10⁸ copies of template, line 7: 80 U/no template, line 8: 80U/1.2×10⁸ copies of template, line 9: 120 U/no template, line 10: 120U/1.2×10⁸ copies of template) (c) during PS-LAMP at 40° C. Cq valueswere plotted with the concentrations of urea (b) and units of Bst 2.0DNA polymerase (d).

FIGS. 19 A-D show the effects of ET SSB and MgSO₄. Fluorescenceintensities were monitored with different amounts of ET SSB (line 1: 0μg/no template, line 2: 0 μg/1.2×10⁸ copies of template, line 3: 0.25μg/no template, line 4: 0.25 μg/1.2×10⁸ copies of template, line 5: 0.5μg/no template, line 6: 0.5 μg/1.2×10⁸ copies of template, line 7: 0.75μg/no template, line 8: 0.75 μg/1.2×10⁸ copies of template) (a) andconcentrations of MgSO₄ (line 1: 0.5 mM/no template, line 2: 0.5mM/1.2×10⁸ copies of template, line 3: 1 mM/no template, line 4: 1mM/1.2×10⁸ copies of template, line 5: 1.5 mM/no template, line 6: 1.5mM/1.2×10⁸ copies of template, line 7: 2 mM/no template, line 8: 2mM/1.2×10⁸ copies of template, line 9: 2.5 mM/no template, line 10: 2.5mM/1.2×10⁸ copies of template) (c) during PS-LAMP at 40° C. Cq valueswere plotted with the amounts of ET SSB (b) and concentrations of MgSO₄(d).

FIGS. 20 A-D show quantitative analysis of PS-LAMP for differenttemplates. The fluorescence intensities were monitored for PS-LAMP at40° C. (a, c) and R-LAMP at 65° C. (b, d) with titrated mers1b (a, b)and mersla (c, d) plasmids.

FIG. 21 shows the scheme of one-step strand displacement (OSD).

FIGS. 22A-F show the effects of ET SSB, Bst 2.0 DNA polymerase and MgSO₄on PS-LAMP at 45° C. for MERS 1b. Fluorescence intensities weremonitored with different amounts of ET SSB (line 1: 0 μg/no template,line 2: 0 μg/1.2×10⁸ copies of template, line 3: 0.5 μg/no template,line 4: 0.5 μg/1.2×10⁸ copies of template, line 5: 1 μg/no template,line 6: 1 μg/1.2×10⁸ copies of template, line 7: 2 μg/no template, line8: 2 μg/1.2×10⁸ copies of template) (a), Bst 2.0 DNA polymerase (line 1:12 U/no template, line 2: 12 U/1.2×10⁸ copies of template, line 3: 16U/no template, line 4: 16 U/1.2×10⁸ copies of template, line 5: 20 U/notemplate, line 6: 20 U/1.2×10⁸ copies of template, line 7: 24 U/notemplate, line 8: 24 U/1.2×10⁸ copies of template) (c) and MgSO₄ (line1: 0 mM/no template, line 2: 0 mM/1.2×10⁸ copies of template, line 3: 1mM/no template, line 4: 1 mM/1.2×10⁸ copies of template, line 5: 2 mM/notemplate, line 6: 2 mM/1.2×10⁸ copies of template, line 7: 3 mM/notemplate, line 8: 3 mM/1.2×10⁸ copies of template, line 9: 4 mM/notemplate, line 10: 4 mM/1.2×10⁸ copies of template) (e) during PS-LAMPat 45° C. DT values were plotted with the amounts of ET SSB (b), Bst 2.0DNA polymerase (d) and concentrations of MgSO₄ (f).

FIGS. 23 A-B show the effects of RecA. Fluorescence intensities weremonitored with a ET SSB (line 1: 0.5 μg/no template, line 2: 0.5μg/1.2×10⁸ copies of template) as a control and different amounts ofRecA (line 3: 1 μg/no template, line 4: 1 μg/1.2×10⁸ copies of template,line 5: 2 μg/no template, line 6: 2 μg/1.2×10⁸ copies of template, line7: 3 μg/no template, line 8: 3 μg/1.2×10⁸ copies of template, line 9: 4μg/no template, line 10: 4 μg/1.2×10⁸ copies of template) during PS-LAMPat 40° C. Cq values were plotted with the amounts of ET SSB and RecA(b). Experimental condition (urea: 1.44 M, Bst 2.0 DNA polymerase: 60 U,MgSO₄: 2 mM).

FIGS. 24A and 24B show PS-LAMP at different temperatures and effect ofMgSO₄ for MERS 1b. Fluorescence intensities for PS-LAMP were monitoredat different temperatures in the normal buffer (line 1: 45° C./notemplate, line 2: 45° C./1.2×10⁸ copies of template, line 3: 50° C./notemplate, line 4: 50° C./1.2×10⁸ copies of template, line 5: 55° C./notemplate, line 6: 55° C./1.2×10⁸ copies of template, line 7: 60° C./notemplate, line 8: 60° C./1.2×10⁸ copies of template) (a). At 45° C.,various MgSO₄ concentrations were tested (line 1: 0 mM/no template, line2: 0 mM/1.2×10⁸ copies of template, line 3: 1 mM/no template, line 4: 1mM/1.2×10⁸ copies of template, line 5: 2 mM/no template, line 6: 2mM/1.2×10⁸ copies of template, line 7: 3 mM/no template, line 8: 3mM/1.2×10⁸ copies of template, line 9: 4 mM/no template, line 10: 4mM/1.2×10⁸ copies of template) in the buffer (8 U of Bst 2.0 DNApolymerase) (b).

FIG. 25 shows selectivity analysis of PS-LAMP for different types ofnon-complementary templates. PS-LAMP with primers specific to MERS 1bwas performed for various templates (blank, MERS 1a, NRP2 and humangDNA) in absence/presence of MERS 1b and the fluorescence intensities ofPS-LAMP were measured after 3 hours at 40° C. in the buffer (1.44 M ofurea, 60 U of Bst 2.0 DNA polymerase, 0.5 μg of ET SSB and 2 mM ofMgSO₄) after 3 hours. The concentration of MERS 1b, MERS 1a, NRP2 andhuman genomic DNA was 500 μg.

FIG. 26A-D show optimization of RTX polymerase. with 12 M Urea: (a)amplification mix, (b), amplification curve (c) and (d) formamideoptimization for zika RNA amplification.

FIG. 27A-C shows final experimental protocol for zika NS5 PS LAMP. Realtime data was collected using formamide at 8%. (a) shows protocol. (b)shows amplification curve. (c) shows picture of zika PS LAMP after 3hour reaction under optimized condition. The LOD is 0.5 fg (˜1000copies).

DETAILED DESCRIPTION

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that theyare not limited to specific synthetic methods or specific recombinantbiotechnology methods unless otherwise specified, or to particularreagents unless otherwise specified, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting.

A. Definitions

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that thethroughout the application, data is provided in a number of differentformats, and that this data, represents endpoints and starting points,and ranges for any combination of the data points. For example, if aparticular data point “10” and a particular data point 15 are disclosed,it is understood that greater than, greater than or equal to, less than,less than or equal to, and equal to 10 and 15 are considered disclosedas well as between 10 and 15. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

A “self-assembly pathway” is a series of reactions autonomously executedby nucleic acid sequences in the execution of hybridized, detectablenucleic acid sequences. The self-assembly pathway comprises assembly, orhybridization, of nucleic acid sequences. In some embodiments, theself-assembly pathway can also comprise one or more disassemblyreactions.

The term “nucleic acid” refers to natural nucleic acids, artificialnucleic acids, analogs thereof, or combinations thereof. Nucleic acidsmay also include analogs of DNA or RNA having modifications to eitherthe bases or the backbone. For example, nucleic acid, as used herein,includes the use of peptide nucleic acids (PNA). The term “nucleicacids” also includes chimeric molecules.

The term “hairpin” as used herein refers to a structure formed byintramolecular base pairing in a single-stranded polynucleotide endingin an unpaired loop (the “hairpin loop”). In various embodiments,hairpins comprise a hairpin loop protected by stems. For example, ahairpin can comprise a first stem region, a hairpin loop region, and asecond stem region. The first and second stem regions can hybridize toeach other and together form a duplex region. Thus, a stem region of ahairpin nucleic acid is a region that hybridizes to a complementaryportion of the same nucleic acid to form the duplex stem of a hairpin.

the term “hairpin loop” refers to a single stranded region that loopsback on itself and is closed by a single base pair.

“Interior loop” and “internal loop,” are used interchangeably and referto a loop closed by two base pairs. The closing base pairs are separateby single stranded regions of zero or more bases. A “bulge loop” is aninterior loop where one of the separated single-stranded regions is zerobases in length and the other is greater than zero bases in length.

An “initiator” is a molecule that is able to initiate the hybridizationof two other nucleic acid sequences. The initiator is also referred toherein as the third nucleic acid sequence, while it facilitates thehybridization of what is referred to herein as the first and secondnucleic acid sequences.

“Monomers” as used herein refers to individual nucleic acid sequences.For example, monomers are referred to herein as a first nucleic acidsequence, a second nucleic acid sequence, or a third nucleic acidsequence, etc.

By “nucleic acid sequence” is meant a nucleic acid which comprises anindividual sequence. When a first, second, or third nucleic acidsequence is referred to, this is meant that the individual nucleotidesof each of the first, second, third, etc., nucleic acid sequence areunique and differ from each other. In other words, the first nucleicacid sequence will differ in nucleotide sequences from the second andthird, etc. There can be multiple nucleic acid sequences with the samesequence. For instance, when a “first nucleic acid sequence” is referredto, this can include multiple copies of the same sequence, all of whichare referred to as a “first nucleic acid sequence.”

Typically, at least two different nucleic acid sequences are used inself-assembly pathways, although three, four, five, six or more may beused. Typically each nucleic acid sequence comprises at least one domainthat is complementary to at least a portion of one other sequence beingused for the self-assembly pathway. Individual nucleic acid sequencesare discussed in more detail below.

The term “domain” refers to a portion of a nucleic acid or proteinsequence. An “input domain” of a nucleic acid or protein sequence refersto a domain that is configured to receive a signal which initiates aphysical and/or chemical change, such as, a for example, aconformational change, of the nucleic acid sequence. In someembodiments, an input domain can be an initiator binding domain, anassembly complement domain, or a disassembly complement domain. An“output domain” of a nucleic acid sequence refers to a domain that isconfigured to confer a signal. For example, the signal can bind acomplementary sequence to an input domain. In some embodiments, anoutput domain is configured to confer a signal to an input domain ofanother nucleic acid sequence. In some embodiments, an output domain canbe, for example, an assembly domain, or a disassembly domain. In someembodiments, an output domain can be present in an initiator.

The term “nucleate” as used herein means to begin a process of, forexample, a physical and/or chemical change at a discrete point in asystem. The term “nucleation” refers to the beginning of physical and/orchemical changes at discrete points in a system.

A “propagation region” as used herein refers to a portion of a domain ofa first nucleic acid sequence that is configured to hybridize to acomplementary second nucleic acid sequence once the toehold of thedomain nucleates at an exposed toehold of the second nucleic acidsequence. The propagation region is configured such that an availablesecondary nucleic acid sequence does not nucleate at the propagationregion; rather, the propagation region hybridizes to the second nucleicacid sequence only after nucleation at the toehold of the same domain.

In some embodiments, nucleic acid sequences can be “metastable.” Thatis, in the absence of an initiator they are kinetically disfavored fromassociating with other nucleic acid sequences comprising complementaryregions.

As used herein, the terms “polymerization” and “assembly” are usedinterchangeably and refer to the association of two or more nucleic acidsequence, or one or more nucleic acid sequences and an initiator, toform a polymer. The “polymer” may comprise covalent bonds, non-covalentbonds or both. For example, in some embodiments a first, second, andthird nucleic acid sequence can hybridize sequentially to form a polymercomprising a three-arm branched junction.

As used herein term “disassembly” refers to the disassociation of aninitiator or at least one nucleic acid sequence.

As used herein “reaction graph” refers to a representation of assembly(and, optionally, disassembly) pathways that can be translated intomolecular executables.

As used herein the terms “flip” and “switch” are used interchangeablyand refer to a change from one state (e.g., accessible) to another state(e.g., inaccessible).

“Kinetically trapped” means that the nucleic acid sequences areinaccessible. In other words, a nucleic acid sequence which is“kinetically trapped” is not available for hybridization. For example, anucleic acid sequence which has formed a hairpin is considered to bekinetically trapped.

As used herein, an “aptamer” is an oligonucleotide that is able tospecifically bind an analyte of interest other than by base pairhybridization. Aptamers typically comprise DNA or RNA or a mixture ofDNA and RNA. Aptamers may be naturally occurring or made by synthetic orrecombinant means. The aptamers are typically single stranded, but mayalso be double stranded or triple stranded. They may comprise naturallyoccurring nucleotides, nucleotides that have been modified in some way,such as by chemical modification, and unnatural bases, for example2-aminopurine. See, for example, U.S. Pat. No. 5,840,867. The aptamersmay be chemically modified, for example, by the addition of a label,such as a fluorophore, or by the addition of a molecule that allows theaptamer to be crosslinked to a molecule to which it is bound. Aptamersare of the same “type” if they have the same sequence or are capable ofspecific binding to the same molecule. The length of the aptamer willvary, but is typically less than about 100 nucleotides.

The term “oligonucleotides,” or “oligos” as used herein refers tooligomers of natural (RNA or DNA) or modified nucleic acid sequences orlinkages, including natural and unnatural deoxyribonucleotides,ribonucleotides, anomeric forms thereof, peptide nucleic acid monomers(PNAs), locked nucleotide acids monomers (LNA), and the like and/orcombinations thereof, capable of specifically binding to asingle-stranded polynucleotide by way of a regular pattern ofsequence-to-sequence interactions, such as Watson-Crick type of basepairing, base stacking, Hoogsteen or reverse Hoogsteen types of basepairing, or the like. Usually nucleic acid sequences are linked byphosphodiester bonds or analogs thereof to form oligonucleotides rangingin size from a few base units, e.g., 8-12, to several tens of baseunits, e.g., 100-200. Suitable oligonucleotides may be prepared by thephosphoramidite method described by Beaucage and Carruthers (TetrahedronLett., 22, 1859-1862, 1981), or by the triester method according toMatteucci, et al. (J. Am. Chem. Soc., 103, 3185, 1981), bothincorporated herein by reference, or by other chemical methods such asusing a commercial automated oligonucleotide synthesizer.Oligonucleotides (both DNA and RNA) may also be synthesizedenzymatically for instance by transcription or strand displacementamplification. Typically, oligonucleotides are single-stranded, butdouble-stranded or partially double-stranded oligos may also be used incertain embodiments of the invention. An “oligo pair” is a pair ofoligos that specifically bind to one another (i.e., are complementary(e.g., perfectly complementary) to one another).

The terms “complementary” and “complementarity” refer tooligonucleotides related by base-pairing rules. Complementarynucleotides are, generally, A and T (or A and U), or C and G. Forexample, for the sequence “5′-AGT-3′,” the perfectly complementarysequence is “3′-TCA-5′.” Methods for calculating the level ofcomplementarity between two nucleic acids are widely known to those ofordinary skill in the art. For example, complementarity may be computedusing online resources, such as, e.g., the NCBI BLAST website(ncbi.nlm.nih.gov/blast/producttable.shtml) and the OligonucleotidesProperties Calculator on the Northwestern University website(basic.northwestem.edu/biotools/oligocalc.html). Two single-stranded RNAor DNA molecules may be considered substantially complementary when thenucleotides of one strand, optimally aligned and with appropriatenucleotide insertions or deletions, pair with at least about 80% of thenucleotides of the other strand, usually at least about 90% to 95%, andmore preferably from about 98 to 100%. Two single-strandedoligonucleotides are considered perfectly complementary when thenucleotides of one strand, optimally aligned and with appropriatenucleotide insertions or deletions, pair with 100% of the nucleotides ofthe other strand. Alternatively, substantial complementarity exists whena first oligonucleotide will hybridize under selective hybridizationconditions to a second oligonucleotide. Selective hybridizationconditions include, but are not limited to, stringent hybridizationconditions. Selective hybridization, or substantially complementaryhybridization, occurs when at least about 65% of the nucleic acidsequences within a first oligonucleotide over a stretch of at least 14to 25 sequences pair with a perfectly complementary sequences within asecond oligonucleotide, preferably at least about 75%, more preferablyat least about 90%. Preferably, the two nucleic acid sequences have atleast 95%, 96%, 97%, 98%, 99% or 100% of sequence identity. See, M.Kanehisa, Nucleic Acids Res. 12, 203 (1984), incorporated herein byreference. For shorter nucleotide sequences selective hybridizationoccurs when at least about 65% of the nucleic acid sequences within afirst oligonucleotide over a stretch of at least 8 to 12 nucleotidespair with a perfectly complementary nucleic acid sequence within asecond oligonucleotide, preferably at least about 75%, more preferablyat least about 90%. Stringent hybridization conditions will typicallyinclude salt concentrations of less than about 1 M, more usually lessthan about 500 mM and preferably less than about 200 mM. Hybridizationtemperatures can be as low as 5° C., and are preferably lower than about30° C. However, longer fragments may require higher hybridizationtemperatures for specific hybridization. Hybridization temperatures aregenerally at least about 2° C. to 6° C. lower than melting temperatures(T_(m)), which are defined below.

As used herein, “two perfectly matched nucleotide sequences” refers to anucleic acid duplex wherein the two nucleotide strands match accordingto the Watson-Crick basepair principle, i.e., A-T and C-G pairs inDNA:DNA duplex and A-U and C-G pairs in DNA:RNA or RNA:RNA duplex, andthere is no deletion or addition in each of the two strands.

The term, “mismatch” refers to a nucleic acid duplex wherein at leastone of the nucleotide base pairs do not form a match according to theWatson-Crick basepair principle. For example, A-C or U-G “pairs” arelined up, which are not capable of forming a basepair. The mismatch canbe in a single set of bases, or in two, three, four, five, or morebasepairs of the nucleic acid duplex.

As used herein, “complementary to each other over at least a portion oftheir sequence” means that at least two or more consecutive nucleotidebase pairs are complementary to each other. For example, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, or more consecutive nucleotide base pairs can becomplementary to each other over the length of the nucleic acidsequence.

As used herein, “substantially hybridized” refers to the conditionsunder which a stable duplex is formed between two nucleic acidsequences, and can be detected. This is discussed in more detail below.

As used herein, a “significant reduction in background hybridization”means that non-specific hybridization, or hybridization betweenunintended nucleic acid sequences, is reduced by at least 80%, morepreferably by at least 90%, even more preferably by at least 95%, stillmore preferably by at least 99%.

By “preferentially binds” it is meant that a specific binding eventbetween a first and second molecule occurs at least 20 times or more,preferably 50 times or more, more preferably 100 times or more, and even1000 times or more often than a nonspecific binding event between thefirst molecule and a molecule that is not the second molecule. Forexample, a capture moiety can be designed to preferentially bind to agiven target agent at least 20 times or more, preferably 50 times ormore, more preferably 100 times or more, and even 1000 times or moreoften than to other molecules in a biological solution. Also, animmobilized binding partner, in certain embodiments, will preferentiallybind to a target agent, capture moiety, or capture moiety/target agentcomplex. While not wishing to be limited by applicants presentunderstanding of the invention, it is believed binding will berecognized as existing when the K_(a) is at 10⁷ l/mole or greater,preferably 10⁸ l/mole or greater. In the embodiment where the capturemoiety is comprised of antibody, the binding affinity of 10⁷ l/mole ormore may be due to (1) a single monoclonal antibody (e.g., large numbersof one kind of antibody) or (2) a plurality of different monoclonalantibodies (e.g., large numbers of each of several different monoclonalantibodies) or (3) large numbers of polyclonal antibodies. It is alsopossible to use combinations of (1)-(3). The differential in bindingaffinity may be accomplished by using several different antibodies asper (1)-(3) above and as such some of the antibodies in a mixture couldhave less than a four-fold difference. For purposes of most embodimentsof the invention an indication that no binding occurs means that theequilibrium or affinity constant K_(a) is 10⁶ l/mole or less. Antibodiesmay be designed to maximize binding to the intended antigen by designingpeptides to specific epitopes that are more accessible to binding, ascan be predicted by one skilled in the art.

The term “sample” in the present specification and claims is used in itsbroadest sense and can be, by non-limiting example, any sample that issuspected of containing a target agent(s) to be detected. It is meant toinclude specimens or cultures (e.g., microbiological cultures), andbiological and environmental specimens as well as non-biologicalspecimens. Biological samples may comprise animal-derived materials,including fluid (e.g., blood, saliva, urine, lymph, etc.), solid (e.g.,stool) or tissue (e.g., buccal, organ-specific, skin, etc.), as well asliquid and solid food and feed products and ingredients such as dairyitems, vegetables, meat and meat by-products, and waste. Biologicalsamples may be obtained from, e.g., humans, any domestic or wildanimals, plants, bacteria or other microorganisms, etc. Environmentalsamples can include environmental material such as surface matter, soil,water (e.g., contaminated water), air and industrial samples, as well assamples obtained from food and dairy processing instruments, apparatus,equipment, utensils, disposable and non-disposable items. These examplesare not to be construed as limiting the sample types applicable to thepresent invention. Those of skill in the art would appreciate andunderstand the particular type of sample required for the detection ofparticular target agents (Pawliszyn, J., Sampling and Sample Preparationfor Field and Laboratory, (2002); Venkatesh Iyengar, G., et al., ElementAnalysis of Biological Samples: Principles and Practices (1998);Drielak, S., Hot Zone Forensics: Chemical, Biological, and RadiologicalEvidence Collection (2004); and Nielsen, D. M., Practical Handbook ofEnvironmental Site Characterization and Ground-Water Monitoring (2005)).

A substance is commonly said to be present in “excess” or “molar excess”relative to another component if that component is present at a highermolar concentration than the other component. Often, when present inexcess, the component will be present in at least a 10-fold molar excessand commonly at 100-1,000,000 fold molar excess. Those of skill in theart would appreciate and understand the particular degree or amount ofexcess preferred for any particular reaction or reaction conditions.Such excess is often empirically determined and/or optimized for aparticular reaction or reaction conditions.

As used herein, “a promoter, a promoter region or promoter element”refers to a segment of DNA or RNA that controls transcription of the DNAor RNA to which it is operatively linked. The promoter region includesspecific sequences that are sufficient for RNA polymerase recognition,binding and transcription initiation. This portion of the promoterregion is referred to as the promoter. In addition, the promoter regionincludes sequences that modulate this recognition, binding andtranscription initiation activity of RNA polymerase. These sequences maybe cis acting or may be responsive to trans acting factors. Promoters,depending upon the nature of the regulation, may be constitutive orregulated.

As used herein, “operatively linked or operationally associated” refersto the functional relationship of nucleic acids with regulatory andeffector sequences of nucleotides, such as promoters, enhancers,transcriptional and translational stop sites, and other signalsequences. For example, operative linkage of DNA to a promoter refers tothe physical and functional relationship between the DNA and thepromoter such that the transcription of such DNA is initiated from thepromoter by an RNA polymerase that specifically recognizes, binds to andtranscribes the DNA. In order to optimize expression and/or in vitrotranscription, it may be necessary to remove, add or alter 5′untranslated portions of the clones to eliminate extra, potentialinappropriate alternative translation initiation (i.e., start) codons orother sequences that may interfere with or reduce expression, either atthe level of transcription or translation. Alternatively, consensusribosome binding sites (see, e.g., Kozak, J. Biol. Chem.,266:19867-19870 (1991)) can be inserted immediately 5′ of the startcodon and may enhance expression. The desirability of (or need for) suchmodification may be empirically determined.

As used herein, “RNA polymerase” refers to an enzyme that synthesizesRNA using a DNA or RNA as the template. It is intended to encompass anyRNA polymerase with conservative amino acid substitutions that do notsubstantially alter its activity.

As used herein, “reverse transcriptase” refers to an enzyme thatsynthesizes DNA using a RNA as the template. It is intended to encompassany reverse transcriptase with conservative amino acid substitutionsthat do not substantially alter its activity.

“Enzymatically produced” refers to the production or secondary ortertiary folding of a nucleic acid by an enzyme rather than by chemicalsynthesis. Enzymatically produced nucleic acids can be made in vitro orin vivo. For example, ribozyme-containing transcription templatescaffolds can be engineered to enable enzymatic co-transcriptionalsynthesis of RNA circuits that can operate without any post-syntheticseparation and re-folding of individual circuit components.

B. Systems, Methods, and Devices

Disclosed herein are systems and methods, as well as the components tobe used to prepare the disclosed systems, devices, and methods. Theseand other materials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a particular nucleic acid sequence is disclosedand discussed and a number of modifications that can be made to a numberof molecules including the nucleic acid sequence are discussed,specifically contemplated is each and every combination and permutationof the nucleic acid sequence and the modifications that are possibleunless specifically indicated to the contrary. Thus, if a class ofmolecules A, B, and C are disclosed as well as a class of molecules D,E, and F and an example of a combination molecule, A-D is disclosed,then even if each is not individually recited each is individually andcollectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F,C-D, C-E, and C-F are considered disclosed. Likewise, any subset orcombination of these is also disclosed. Thus, for example, the sub-groupof A-E, B-F, and C-E would be considered disclosed. This concept appliesto all aspects of this application including, but not limited to, stepsin methods of making and using the disclosed compositions. Thus, ifthere are a variety of additional steps that can be performed it isunderstood that each of these additional steps can be performed with anyspecific embodiment or combination of embodiments of the disclosedmethods.

1. Thermophilic Strand-Displacing Polymerase

Strand-displacing polymerases are a crucial component of isothermalamplification (IA) reactions, DNA amplification techniques that, unlikePCR, don't require thermal cycling. Because they simplify equipmentneeds and are often faster and less prone to inhibition than PCR, thesetechniques are appealing for nucleic acid-based point of care detection.While pre-reaction heating is useful in isothermal amplification (IA)for cell lysis, contaminant denaturation, and template denaturation forimproved primer binding, there is currently no thermostable polymerasedemonstrated for multiple IA mechanisms that can survive these hightemperatures, complicating point of care workflow. Additionally,polymerase engineering of strand-displacing polymerases has been limitedto in silico approaches due to the inherent challenges in evolvingmesophilic polymerases and selecting for strand displacement.

Disclosed herein is isothermal compartmentalized self-replication(IsoCSR), an isothermal emulsion-based directed evolution methodanalogous to CSR. Using an algorithm-optimized shuffled library ofexonuclease-deficient polymerases from Geobacillus stearothermophilus(Bst LF) and Thermus aquaticus (Klentaq), IsoCSR was demonstrated byevolving a thermostable strand-displacing polymerase capable of hightemperature IA methods like loop-mediated isothermal amplification androlling circle amplification (LAMP and RCA), even after incubation attemperatures as high as 95° C. The polymerase outperforms Bst LFpolymerase, the high temperature isothermal polymerase of choice, in RCAand performs similarly in LAMP reactions. IsoCSR is essential forevolving other useful characteristics for point of care applications,such as enhanced inhibitor tolerance in unprocessed samples.

Although Bst LF works at higher temperatures, it is not trulythermostable at temperatures that may be required for DNA denaturationand ‘hot start’ LAMP. There have been reports of two thermostablepolymerases that could potentially be included in a denaturation step inIA reactions such as LAMP: OmniAmp, a viral polymerase from PyroPhage3173 with DNA polymerase and reverse transcriptase activities (Chander2014), and SD Polymerase, a mutant of the well-known Thermus aquaticus(Taq) polymerase (Ignatov 2014). However, neither of these polymeraseshave been validated for hot start LAMP, either due to the lack ofsufficient thermostability to survive denaturation steps (OmniAmp), orinsufficient strand displacement activity for LAMP (SD Polymerase).

Directed polymerase evolution using compartmentalized self-replication(CSR) and other methods have previously been used to identify sequencevariants of DNA and RNA polymerases that have altered phenotypes such asincreased thermostability, incorporation of unnatural or modified bases,reverse transcription, orthogonal promoter recognition, and resistanceto enzymatic inhibitors (Ghadessy 2001; Baar 2011; Chen 2014; Meyer2015; Ellefson 2016). Recently, an isothermal CSR selection was used toevolve the phi29 polymerase, but this selection required cumbersomefreezing and thawing cycles for cell lysis that may limit theacquisition of thermostability or other novel phenotypes. In addition,the use of random primers resulted in off-target E. coli genomeamplification, limiting the selection's utility and efficiency(Povilaitis 2016). A more robust isothermal compartmentalizedself-replication (IsoCSR) method has been developed for engineeringthermostable strand-displacing polymerases. IsoCSR retains theemulsion-based linkage of genotype and phenotype that was established inthermal cycling CSR, but replaces the in emulsio PCR step withhyperbranched rolling circle amplification (hbRCA) of supercoiledplasmid DNA. This innovation necessitates that a polymerase haveexcellent strand displacement activity in order to self-amplify itsgene.

IsoCSR was used to combine the robust strand displacement capability ofBst LF with the extreme thermostability found in Klentaq. Thesedistantly related enzymes were recoded to ensure maximal overlap, ashuffled library was created, and a thermostable chimeric polymerase wasselected that enabled one pot, hot start LAMP. Strand displacementseemed to arise from a relatively short Bst insertion into a Klentaqbackbone that can stabilize the ‘thumb’ domain common to DNApolymerases.

Therefore, disclosed herein is a non-naturally occurring thermostablepolymerase, wherein thermostable polymerase is characterized byincreased temperature stability in the range of 70° C. to 100° C.,increased strand displacement capability, increased processivity, or acombination thereof compared with a wild type large fragment Bacillusstearothermophilus (Bst LF) polymerase. The polymerase is also morethermostable than Bst 2.0. The polymerase is capable of both isothermalamplification, such as LAMP and hyperbranched rolling circleamplification (RCA) from supercoiled plasmids without the use of nickingendonucleases. It is also useful with strand displacement amplification(SDA), polymerase spiral reaction (PSR), or helicase dependentamplification (HDA). The polymerase can also be capable of replicatingDNA in a polymerase chain reaction (PCR).

By “increased temperature stability” is meant that the polymerase is 2,3, 4, 5, 6, 7, 8, 9, 10, or 100 ore more times stable at the range of 70to 100° C. than other polymerases. By “increased strand displacementcapability” is meant that the polymerase disclosed herein is 10, 20, 30,40, 50, 60, 70, 80, 90, or 100% or more capable of strand displacementthan other polymerases. By “increased processivity” is meant that thepolymerase disclosed herein is 10, 20, 30, 40, 50, 60, 70, 80, 90, or100% or more processive than other polymerases.

The thermostable polymerase disclosed herein can be heated to above 60,65, 70, 75, 80, 85, 90, 95, or 100° C. or higher without significantdenaturation. Amplification can also occur at elevated temperatures,such as above 65° C.

As described in Example 1, the thermostable polymerase can comprisesequences from at least Bst LF and Klentaq polymerase. For example,disclosed is a polymerase which is 90, 91, 92, 93, 94, 95, 96, 97, or99% or more identical to SEQ ID NO: 1. Also disclosed is a nucleic acidencoding SEQ ID NO: 1. For example, disclosed herein is the nucleic acidof SEQ ID NO: 2, which encodes a thermostable polymerase. This specificthermostable polymerase is referred to herein as v5.9. Also disclosed isa host cell comprising the nucleic acid encoding the polymerasedisclosed herein.

The thermostable polymerase disclosed herein can be stored in a storagebuffer, or a reaction buffer. For example, the buffer can comprise atemperature dependent inhibitor of polymerase activity. The polymerasecan substantially lack 3′ to 5′ exonuclease activity.

Also disclosed herein is a method of identifying a non-naturallyoccurring thermostable polymerases, the method comprising: a) providinga pool of nucleic acids comprising nucleic acid members each encodingnon-naturally occurring, potential thermostable polymerases; b)subdividing the pool of nucleic acid members into cellular compartmentsby transformation into a bacterial host, such that each cell comprises anucleic acid member; c) expressing the nucleic acid member in the cellcompartment to form a potential thermostable polymerase encoded by thenucleic acid member; d) subdividing the pool of bacterial cells intocompartments, such that each compartment contains a single cell with asingle nucleic acid member and encoded polymerase e) subjecting the poolof nucleic acid members to thermal denaturation and isothermalamplification conditions, such that the nucleic acid member may beprocessed by the a thermostable polymerase encoded by said nucleic acidmember; and f) detecting processing of the nucleic acid member by athermostable polymerase encoded by said nucleic acid member, therebyidentifying a thermostable polymerase.

The pool of nucleic acids can be created by gene shuffling, error-pronePCR, or site-saturation mutagenesis. For example, gene shuffling cantake place with nucleic acid encoding one or more known polymerases.This can be seen in Example 1. The polymerases can include, but are notlimited to, Bst LF and Klentaq, for example. Thermal denaturation cantake place at 65, 70, 75, 80, 85, 90, 95, or 100° C. or higher.

The isothermal amplification to which the polymerase can be subjectedcan be, but is not limited to, hyperbranched rolling circleamplification, recombinase polymerase amplification, or loop-mediatedisothermal amplification. Amplification can also be by polymerase chainreaction (PCR). In one example, the contents of each compartment are notin contact with the contents of other compartments. The processing ofthe nucleic acid member can result in one copy of said nucleic acidmember. The processing of the nucleic acid member can also result inmore than one copy of said nucleic acid member. In one example, thenumber of copies of the nucleic acid member can be proportional to theactivity of the thermostable polymerase.

Processing of nucleic acids can be detected by assaying the copy numberof the nucleic acid member. For example, processing can be detected byassaying the presence of a tag on the nucleic acid member. Processingcan also be detected by determining thermostable polymerase activity.

In one example, the step of expressing the nucleic acid member to formthe thermostable polymerase encoded by said nucleic acid member can becarried out by in vitro transcription and translation. The step ofexpressing the nucleic acid member to form the thermostable polymeraseencoded by said nucleic acid member can be carried out by in vivotranscription and translation in an expression host cell. The expressionhost cell can be a bacterial cell.

The compartments for carrying out the assay can comprise aqueouscompartments of a water-in-oil emulsion. In one example, the non-aqueousportion of emulsion mix is 73% Tegosoft DEC, 7% AbilWE09, and 20%mineral oil. with an oil phase and a surfactant comprising Span80,Tween80, and TritonX100. The surfactant can comprise AbilWE09.

2. Isothermal Amplification Using Phosphorothioated Loop MediatedIsothermal Amplification (PS-LAMP)

Loop-mediated isothermal amplification (LAMP) is an extremely powerfultool for the detection of nucleic acids with high sensitivity andspecificity. LAMP shows best performance at around 65° C., which isrelatively high to be easily applied to point-of-care-testing (POCT).Disclosed herein is a phosphorothioated LAMP (PS-LAMP) working at lowtemperature by providing a more efficient amplification path.PS-modifications enabled efficient self-folding at the termini togenerate more loops where intact inner primers bind and extend. Byoptimizing several factors such as urea, Bst 2.0 DNA polymerase,single-stranded DNA binding protein (SSB) and MgSO₄, comparablesensitivity and selectivity with a regular LAMP (R-LAMP) at 65° C. wereachieved with a PS-LAMP at 40° C. As the novel PS-LAMP system isperformed at around physiological temperature, it has a great potentialin applications to hand-held or POCT diagnostic devices.

By incorporating the PS-THSP mechanism into a regular LAMP system, LAMPthat performs well at low temperature has been made (Example 2). Aphosphorothioate (PS) modification is incorporated into a part of innerprimer's DNA backbone, leading to an increased self-folding of terminalhairpins. It can produce more loop sites where other inner primers canbind to and finally result in more efficient amplification. Byoptimizing conditions to accelerate the self-folding, the sensitivity ofPS-LAMP at 40° C. was comparable with that of a regular LAMP at optimaltemperature.

The PS-modified DNA can also display enhanced stability againstdegradation by various nucleases that may be present in biologicalsamples. As the novel PS-LAMP system is performed at aroundphysiological temperature, it can be used in hand-held or point-of-care(POC) diagnostic devices.

Regarding LAMP in particular, it is a powerful isothermal nucleic acidamplification technique that can generate ˜109 copies from less than 10copies of template DNA within an hour or two. Unfortunately, while theamplification reactions are extremely powerful, quantitative detectionof LAMP products has remained analytically challenging. LAMP can beconducted with two, three, four, five, or six primers, for example.OSD-LAMP can be used with 2 primers (FIP+BIP) and also 3 primers(FIP+BIP+F3 and FIP+BIP+B3). 2 as well as 3-primer OSD-LAMP assays canalso be used The five primer LAMP system disclosed herein, and depictedin FIG. 1, is ultra-fast, sensitive, and a highly selective.

The 4-primer LAMP is the basic form of LAMP that was originallydescribed for isothermal nucleic acid amplification. The system iscomposed of two loop-forming inner primers FIP and BIP and two outerprimers F3 and B3 whose primary function is to displace the DNA strandsinitiated from the inner primers thus allowing formation of the loopsand strand displacement DNA synthesis. Subsequently 6-primer LAMP wasreported that incorporated 2 additional primers, LF and LB, that bind tothe loop sequences located between the F1/F1c and F2/F2c priming sitesand the B1/B1c and B2/B2c priming sites. Addition of both loop primerssignificantly accelerated LAMP. The 5-primer LAMP has been describedherein, wherein the 4 LAMP primers (F3, B3, FIP and BIP) are used inconjunction with only one of the loop primers (either LF or LB). Thisallows the accelerated amplification afforded by the loop primer whileusing the other LAMP loop (not bound by the loop primer) forhybridization to loop-specific OSD probe. This innovation allows forhigh-speed LAMP operation while performing real-time sequence-specificsignal transduction.

Therefore, disclosed herein is a method of using PS-LAMP comprisingamplifying a nucleic acid, the method comprising exposing a targetnucleic acid to a buffer solution comprising a polymerase and at leastfour primers, wherein at least one of the four primers comprises aphosphorothioated nucleotide; and amplifying the target nucleic acidusing an isothermal amplification reaction, wherein the isothermalamplification reaction produces at least one loop product, wherein atleast part of the single-stranded portion of the loop product representsthe target nucleic acid.

In the PS-LAMP method disclosed herein, the loop product is exposed to astrand displacement reporter, wherein the strand displacement reportercomprises single-stranded and double-stranded nucleic acid, and furtherwherein a portion of the single-stranded nucleic acid of the stranddisplacement reporter is complementary to at least a portion of thesingle-stranded nucleic acid of the loop product representing the targetnucleic acid, and allowing the loop product and the strand displacementreporter to interact, wherein interaction between the stranddisplacement reporter and the target nucleic acid portion of the loopproduct produces a detectable signal, wherein the signal indicates thepresence of the target nucleic acid.

The isothermal amplification reaction can take place at 20, 25, 30, 35,40, 45, 50, or 55° C. In one specific embodiment, the isothermalreaction can take place around 40° C.

The buffer can comprise various components which have been optimized forPS-LAMP. For example, urea can be present in the buffer at aconcentration of 1.3-1.6 M, specifically 1.44 M. The buffer can alsocomprise a DNA polymerase, such as Bst, specifically Bst 2.0. Bst 2.0can be present at a concentration of 35, 30, 45, 50, 55, 60, 65, or moreunits (U). The buffer can comprise MgSO₄. MgSO₄ can be present at aconcentration of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,2.1, 2.2, 2.3, 2.4, or 2.5 or more mM. The buffer can also comprise aSingle-Stranded Binding (SSB) protein. SSB can be present at aconcentration of about 0.2 to 0.7 μg, for example about 0.5 μg. Specificexamples of buffers can be found in Example 2.

In the method disclosed herein, the strand displacement reporter can beone step toehold displacement (OSD) reporter. The target nucleic acidcan be RNA or DNA. Four, five, or six primers can be used with theisothermal amplification reaction.

FIG. 16 shows a reaction schematic for PS-LAMP. For example, the methodcan comprise using at least one forward inner primer (FIP), at least onebackward inner primer (BIP), at least one forward outer primer (FOP),and at least one backward outer primer (BOP). At least one FIP comprisesat least one phosphorothioated nucleotide. At least one BIP can compriseat least one phosphorothioated nucleotide. Both a FIP and a BIP cancomprise at least one phosphorothioated nucleotide. For example, two ormore nucleotides of at least one primer are phosphorothioated. 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or morenucleotides of a primer can be phosphorothioated. The phosphorothioatednucleotides can be at either the N-terminal or the C-terminal of theprimer.

Amplification of the target nucleic acid takes place in real time. Manyexamples of real-time amplification are known to those of skill in theart. One of skill in the art could therefore readily ascertain areal-time method for use with the invention disclosed herein.

Also disclosed is a method of diagnosing a subject with a disease, themethod comprising carrying out the method of amplification describedherein, wherein the presence of a target nucleic acid indicates thepresence of a disease in the subject.

In one example, multiple target nucleic acids can be amplifiedsimultaneously. The primers can bind a primer binding region of thetarget nucleic acid, as shown in FIG. 16. The method can take place in asingle vessel, also referred to herein as “one pot” amplification.

Disclosed herein is a kit for amplifying nucleic acids, wherein the kitcomprises DNA polymerase, and at least four distinct primers, wherein atleast one of the primers is phosphorothioated. The kit can furthercomprise a buffer solution.

LAMP can be carried out using DNA or RNA (RT-LAMP). LAMP can amplifynucleic acids from a wide variety of samples. These include, but notlimited to, bodily fluids (including, but not limited to, blood, urine,serum, lymph, saliva, anal and vaginal secretions, perspiration andsemen, of virtually any organism, with mammalian samples being preferredand human samples being particularly preferred); environmental samples(including, but not limited to, air, agricultural, water and soilsamples); plant materials; biological warfare agent samples; researchsamples (for example, the sample may be the product of an amplificationreaction, for example general amplification of genomic DNA); purifiedsamples, such as purified genomic DNA, RNA, proteins, etc.; raw samples(bacteria, virus, genomic DNA, etc.); as will be appreciated by those inthe art, virtually any experimental manipulation may have been done onthe sample. Specifically, it is noted that the polymerases disclosedherein can be used to amplify Zika Virus. Some embodiments utilize siRNAand microRNA as target sequences (Zhang et al., J Cell Physiol. (2007)210(2):279-89; Osada et al., Carcinogenesis. (2007) 28(1):2-12; andMattes et al., Am J Respir Cell MoI Biol. (2007) 36(1):8-12, each ofwhich is incorporated herein by reference in its entirety).

Some embodiments utilize nucleic acid samples from stored (e.g. frozenand/or archived) or fresh tissues. Paraffin-embedded samples are ofparticular use in many embodiments, as these samples can be very useful,due to the presence of additional data associated with the samples, suchas diagnosis and prognosis. Fixed and paraffin-embedded tissue samplesas described herein refers to storable or archival tissue samples. Mostpatient-derived pathological samples are routinely fixed andparaffin-embedded to allow for histological analysis and subsequentarchival storage.

The target analytes can be nucleic acids. A nucleic acid of the presentinvention, whether referring to the target nucleic acid or the stranddisplacement reporter, will generally contain phosphodiester bonds (forexample in the case of the target sequences), although in some cases,nucleic acid analogs are included that may have alternate backbones,comprising, for example, phosphoramide (Beaucage et al., Tetrahedron(1993) 49(10):1925 and references therein; Letsinger, J. Org. Chem.(1970) 35:3800; Sprinzl et al., Eur. J. Biochem. (1977) 81:579;Letsinger et al., Nucl. Acids Res. (1986) 14:3487; Sawai et al, Chem.Lett. (1984) 805; Letsinger et al., J. Am. Chem. Soc. (1988) 110:4470;and Pauwels et al., Chemica Scripta (1986) 26:141), phosphorothioate(Mag et al., Nucleic Acids Res. (1991) 19:1437; and U.S. Pat. No.5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc. (1989)111:2321, O-methylphophoroamidite linkages (see Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress), and peptide nucleic acid backbones and linkages (see Egholm, J.Am. Chem. Soc. (1992)114:1895; Meier et al., Chem. Int. Ed. Engl. (1992)31:1008; Nielsen, Nature, (1993) 365:566; Carlsson et al., Nature (1996)380:207, all of which are incorporated herein by reference in theirentirety). Other analog nucleic acids include those with bicyclicstructures including locked nucleic acids, Koshkin et al., J. Am. Chem.Soc. (1998) 120:13252 3); positive backbones (Denpcy et al., Proc. Natl.Acad. Sci. USA (1995) 92:6097; non-ionic backbones (U.S. Pat. Nos.5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi etal., Angew. Chem. Intl. Ed. English (1991) 30:423; Letsinger et al., J.Am. Chem. Soc. (1988) 110:4470; Letsinger et al., Nucleoside &Nucleotide (1994) 13:1597; Chapters 2 and 3, ASC Symposium Series 580,Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic &Medicinal Chem. Lett. (1994) 4:395; Jeffs et al., J. Biomolecular NMR(1994) 34:17; Xu et al., Tetrahedron Lett. (1996) 37:743) and 7non-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or morecarbocyclic sugars are also included within the definition of nucleicacids (see Jenkins et al., Chem. Soc. Rev. (1995) pp 169-176). Severalnucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997page 35. All of these references are herein expressly incorporated byreference. These modifications of the ribose-phosphate backbone may bedone to facilitate the addition of labels or other moieties, to increaseor decrease the stability and half-life of such molecules inphysiological environments, etc.

3. Uses

The methods, products, and devices disclosed herein can be used formultiple applications. Detection and identification of virtually anynucleic acid sequence, or non-nucleic acid sequence, can beaccomplished. For example, the presence of specific viruses,microorganisms and parasites can be detected. For example, Zika Viruscan be amplified. Genetic diseases can also be detected and diagnosed,either by detection of sequence variations (mutations) which cause orare associated with a disease or are linked (Restriction Fragment LengthPolymorphisms or RFLP's) to the disease locus. Sequence variations whichare associated with, or cause, cancer, can also be detected. This canallow for both the diagnosis and prognosis of disease. For example, if abreast cancer marker is detected in an individual, the individual can bemade aware of their increased likelihood of developing breast cancer,and can be treated accordingly. The methods and devices disclosed hereincan also be used in the detection and identification of nucleic acidsequences for forensic fingerprinting, tissue typing and for taxonomicpurposes, namely the identification and speciation of microorganisms,flora and fauna.

The methods and devices disclosed herein have applications in clinicalmedicine, veterinary science, aquaculture, horticulture and agriculture.The methods and devices can also be used in maternity and paternitytesting, fetal sex determination, and pregnancy tests.

4. Devices

Disclosed herein are devices for detection of a target nucleic acid,wherein the device comprises: a) an amplification unit, wherein saidamplification unit amplifies the target nucleic acid via an isothermalamplification reaction); b) a transducer, wherein said transducercomprises strand displacement reporters, wherein said stranddisplacement reporters interact with the target nucleic acidamplification product of step a); and c) a signal output unit, whichdisplays the detectable signal of step b).

The amplification unit is the portion of the device where amplificationof a nucleic acid takes place. This can be via the LAMP methodsdisclosed herein, for example. The signal output unit detects the signalfrom the strand displacement reporter. The signal output unit can bepart of a computer system, and the signal can be displayed on a monitor.The resulting signal can also be used in a computer processor to compareit to other results or databases, and the results can be displayed.Computer systems and computer readable media are discussed in moredetail below.

The amplification unit, transducer, and signal output unit can be in asingle device, and can be in fluid communication with each other. Forexample, amplification and detection can all take place in the same wellof a microfluidics device. Furthermore, amplification and detection cantake place simultaneously, and detection can occur in “real time.”

The device can also comprise a heater. Because the amplification anddetection reactions may require a temperature above room temperature, aheat source is contemplated herein. Heat sources may include, but arenot limited to, contacting and non-contacting sources, as known in theart. In one embodiment, the heat source may comprise an optical heatingdevice. For example, the optical device may comprise a defocused laserthat is directed at an underside of the device. For example, heating maybe achieved by using an 808 nm infra-red laser diode module (e.g.,icetec-UK) operating at approximately 150 mW directed onto the device.The power of the laser may be controlled through an n-channel powerMOSFET gated by a logic optocoupler driven by pulse width modulated(PWM) signal from a microcontroller (e.g., Fox LP3500, RabbitSemiconductor, Davis, Calif.).

To provide temperature control, the controller may be programmed with amodified proportional-integral control routine using feedback from thepyrometer. The pyrometer feedback may be received by the microcontrollerafter a calibration correction is applied. To perform opticaltemperature detection, the sample may be illuminated obliquely, forexample, by a high intensity light source having a selected wavelength.In one embodiment, the light source may comprise a blue light emittingdiode (LED) that emits light at a wavelength selected within the rangebetween about 450 nm to about 475 nm (e.g., approximately 470 nm). Anexample of an LED light source capable of this illumination is HLMP CB28STD00, manufactured by Agilent Technologies, Santa Clara, Calif. Heatingmay also be achieved by other methods such as by chemical exothermicreactions or by using the computer's CPU-generated heat, or heatingspecific metals with batteries, etc.

5. Detection

Detection systems are known in the art, and include optical assays(including fluorescence and chemiluminescent assays), enzymatic assays,radiolabelling, surface plasmon resonance, magnetoresistance, cantileverdeflection, surface plasmon resonance, etc. In some embodiments, OSDreporter can be used in additional assay technologies, for example, asdescribed in 2006/0068378, hereby incorporated by reference, the OSDreporter can serve as a linker between light scattering particles suchas colloids, resulting in a color change in the presence of the OSDreporter.

In some embodiments, the strand displacement reporters of the inventionare attached to solid supports for detection. For example, stranddisplacement reporters can be attached to beads for subsequent analysis.Similarly, bead arrays as described below may be used.

In one embodiment, the present invention provides arrays, each arraylocation comprising at a minimum a covalently attached stranddisplacement reporter, also referred to herein as a “capture probe”. By“array” herein is meant a plurality of nucleic acid probes (OSDreporters, for example) in an array format; the size of the array willdepend on the composition and end use of the array. Generally, the arraywill comprise from two to as many as 100,000 or more reporters,depending on the size of the electrodes, as well as the end use of thearray. Preferred ranges are from about 2 to about 10,000, with fromabout 5 to about 1000 being preferred, and from about 10 to about 100being particularly preferred. In some embodiments, the compositions ofthe invention may not be in array format; that is, for some embodiments,compositions comprising a single capture probe may be made as well. Inaddition, in some arrays, multiple substrates may be used, either ofdifferent or identical compositions. Thus, for example, large arrays maycomprise a plurality of smaller substrates. Nucleic acids arrays areknown in the art, and can be classified in a number of ways; bothordered arrays (e.g. the ability to resolve chemistries at discretesites), and random arrays (e.g. bead arrays) are included. Orderedarrays include, but are not limited to, those made usingphotolithography techniques (Affymetrix GeneChip™), spotting techniques(Synteni and others), printing techniques (Hewlett Packard and Rosetta),origami pads, paperfluidics, electrode arrays, three dimensional “gelpad” arrays, etc. Liquid arrays may also be used.

By “substrate” or “solid support” or other grammatical equivalentsherein is meant any material that can be modified to contain discreteindividual sites appropriate for the attachment or association ofnucleic acids. The substrate can comprise a wide variety of materials,as will be appreciated by those in the art. including, but not limitedto glass, plastics, polymers, metals, metalloids, ceramics, organics,etc. When the solid support is a bead, a wide variety of substrates arepossible, including magnetic materials, glass, silicon, dextrans,plastics, etc.

In another aspect of the invention, a fluidic is used to automate themethodology described in this invention. See for example U.S. Pat. No.6,942,771, herein incorporated by reference for components including butnot limited to cartridges, devices, pumps, wells, reaction chambers, anddetection chambers.

The devices of the invention can comprise liquid handling components,including components for loading and unloading fluids at each station orsets of stations. The liquid handling systems can include roboticsystems comprising any number of components. In addition, any or all ofthe steps outlined herein may be automated; thus, for example, thesystems may be completely or partially automated.

As will be appreciated by those in the art, there are a wide variety ofcomponents which can be used, including, but not limited to, one or morerobotic arms; plate handlers for the positioning of microplates; holderswith cartridges and/or caps; automated lid or cap handlers to remove andreplace lids for wells on non-cross contamination plates; tip assembliesfor sample distribution with disposable tips; washable tip assembliesfor sample distribution; 96 well loading blocks; cooled reagent racks;microtitler plate pipette positions (optionally cooled); stacking towersfor plates and tips; and computer systems.

Fully robotic or microfluidic systems include automated liquid-,particle-, cell- and organism-handling including high throughputpipetting to perform all steps of screening applications. This includesliquid, particle, cell, and organism manipulations such as aspiration,dispensing, mixing, diluting, washing, accurate volumetric transfers;retrieving, and discarding of pipet tips; and repetitive pipetting ofidentical volumes for multiple deliveries from a single sampleaspiration. These manipulations are cross-contamination-free liquid,particle, cell, and organism transfers. This instrument performsautomated replication of microplate samples to filters, membranes,and/or daughter plates, high-density transfers, full-plate serialdilutions, and high capacity operation.

Chemically derivatized particles, plates, cartridges, tubes, magneticparticles, or other solid phase matrix with specificity to the assaycomponents can also used. The binding surfaces of microplates, tubes orany solid phase matrices include non-polar surfaces, highly polarsurfaces, modified dextran coating to promote covalent binding, antibodycoating, affinity media to bind fusion proteins or peptides,surface-fixed proteins such as recombinant protein A or G, nucleotideresins or coatings, and other affinity matrix are useful in thisinvention.

Platforms for multi-well plates, multi-tubes, holders, cartridges,minitubes, deep-well plates, microfuge tubes, cryovials, square wellplates, fitters, chips, optic fibers, beads, and other solid-phasematrices or platform with various volumes can be accommodated on anupgradable modular platform for additional capacity. This modularplatform includes a variable speed orbital shaker, and multi-positionwork decks for source samples, sample and reagent dilution, assayplates, sample and reagent reservoirs, pipette tips, and an active washstation.

Interchangeable pipet heads (single or multi-channel) with single ormultiple magnetic probes, affinity probes, or pipetters roboticallymanipulate the liquid, particles, cells, and organisms can be used.Multi-well or multi-tube magnetic separators or platforms manipulateliquid, particles, cells, and organisms in single or multiple sampleformats.

The instrumentation can include a detector, which can be a wide varietyof different detectors, depending on the labels and assay. In apreferred embodiment, useful detectors include a microscope(s) withmultiple channels of fluorescence; plate readers to provide fluorescent,electrochemical and/or electrical impedance analyzers, ultraviolet andvisible spectrophotometry detection with single and dual wavelengthendpoint and kinetics capability, fluroescence resonance energy transfer(FRET), luminescence, quenching, two-photon excitation, and intensityredistribution; CCD cameras to capture and transform data and imagesinto quantifiable formats; and a computer workstation.

These instruments can fit in a sterile laminar flow or fume hood, or areenclosed, self-contained systems, for cell culture growth andtransformation in multi-well plates or tubes and for hazardousoperations. The living cells may be grown under controlled growthconditions, with controls for temperature, humidity, and gas for timeseries of the live cell assays. Automated transformation of cells andautomated colony pickers may facilitate rapid screening of desiredcells. Flow cytometry or capillary electrophoresis formats can be usedfor individual capture of magnetic and other beads, particles, cells,and organisms.

The flexible hardware and software allow instrument adaptability formultiple applications. The software program modules allow creation,modification, and running of methods. The system diagnostic modulesallow instrument alignment, correct connections, and motor operations.The customized tools, labware, and liquid, particle, cell and organismtransfer patterns allow different applications to be performed. Thedatabase allows method and parameter storage. Robotic and computerinterfaces allow communication between instruments.

The robotic apparatus can include central processing unit whichcommunicates with a memory and a set of input/output devices (e.g.,keyboard, mouse, monitor, printer, etc.) through a bus. Again, asoutlined below, this may be in addition to or in place of the CPU forthe multiplexing devices of the invention. The general interactionbetween a central processing unit, a memory, input/output devices, and abus is known in the art. Thus, a variety of different procedures,depending on the experiments to be run, are stored in the CPU memory.These robotic fluid handling systems can utilize any number of differentreagents, including buffers, reagents, samples, washes, assay componentssuch as label probes, etc.

6. Hybridization/Selective Hybridization

The term hybridization typically means a sequence driven interactionbetween at least two nucleic acid molecules, such as a primer or a probeand a gene. Sequence driven interaction means an interaction that occursbetween two nucleotides or nucleotide analogs or nucleotide derivativesin a nucleotide specific manner. For example, G interacting with C or Ainteracting with T are sequence driven interactions. Typically sequencedriven interactions occur on the Watson-Crick face or Hoogsteen face ofthe nucleotide. The hybridization of two nucleic acids is affected by anumber of conditions and parameters known to those of skill in the art.For example, the salt concentrations, pH, and temperature of thereaction all affect whether two nucleic acid molecules will hybridize.

Parameters for selective hybridization between two nucleic acidmolecules are well known to those of skill in the art. For example, insome embodiments selective hybridization conditions can be defined asstringent hybridization conditions. For example, stringency ofhybridization is controlled by both temperature and salt concentrationof either or both of the hybridization and washing steps. For example,the conditions of hybridization to achieve selective hybridization mayinvolve hybridization in high ionic strength solution (6×SSC or 6×SSPE)at a temperature that is about 12-25° C. below the Tm (the meltingtemperature at which half of the molecules dissociate from theirhybridization partners) followed by washing at a combination oftemperature and salt concentration chosen so that the washingtemperature is about 5° C. to 20° C. below the Tm. The temperature andsalt conditions are readily determined empirically in preliminaryexperiments in which samples of reference DNA immobilized on filters arehybridized to a labeled nucleic acid of interest and then washed underconditions of different stringencies. Hybridization temperatures aretypically higher for DNA-RNA and RNA-RNA hybridizations. The conditionscan be used as described above to achieve stringency, or as is known inthe art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989;Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which is hereinincorporated by reference for material at least related to hybridizationof nucleic acids). A preferable stringent hybridization condition for aDNA:DNA hybridization can be at about 68° C. (in aqueous solution) in6×SSC or 6×SSPE followed by washing at 68° C. Stringency ofhybridization and washing, if desired, can be reduced accordingly as thedegree of complementarity desired is decreased, and further, dependingupon the G-C or A-T richness of any area wherein variability is searchedfor. Likewise, stringency of hybridization and washing, if desired, canbe increased accordingly as homology desired is increased, and further,depending upon the G-C or A-T richness of any area wherein high homologyis desired, all as known in the art.

Another way to define selective hybridization is by looking at theamount (percentage) of one of the nucleic acids bound to the othernucleic acid. For example, in some embodiments selective hybridizationconditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid isbound to the non-limiting nucleic acid. Typically, the non-limitingprimer is in for example, 10 or 100 or 1000 fold excess. This type ofassay can be performed at under conditions where both the limiting andnon-limiting primer are for example, 10 fold or 100 fold or 1000 foldbelow their k_(d), or where only one of the nucleic acid molecules is 10fold or 100 fold or 1000 fold or where one or both nucleic acidmolecules are above their k_(d).

Another way to define selective hybridization is by looking at thepercentage of primer that gets enzymatically manipulated underconditions where hybridization is required to promote the desiredenzymatic manipulation. For example, in some embodiments selectivehybridization conditions would be when at least about, 60, 65, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer isenzymatically manipulated under conditions which promote the enzymaticmanipulation, for example if the enzymatic manipulation is DNAextension, then selective hybridization conditions would be when atleast about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100percent of the primer molecules are extended. Preferred conditions alsoinclude those suggested by the manufacturer or indicated in the art asbeing appropriate for the enzyme performing the manipulation.

Just as with homology, it is understood that there are a variety ofmethods herein disclosed for determining the level of hybridizationbetween two nucleic acid molecules. It is understood that these methodsand conditions may provide different percentages of hybridizationbetween two nucleic acid molecules, but unless otherwise indicatedmeeting the parameters of any of the methods would be sufficient. Forexample if 80% hybridization was required and as long as hybridizationoccurs within the required parameters in any one of these methods it isconsidered disclosed herein.

It is understood that those of skill in the art understand that if acomposition or method meets any one of these criteria for determininghybridization either collectively or singly it is a composition ormethod that is disclosed herein.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary and arenot intended to limit the disclosure. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.), butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

C. Examples 1. Example 1: Evolution of a Thermophilic Strand-DisplacingPolymerase Using Isothermal Compartmentalized Self Replication

a) Materials and Methods

(1) Strains, Primers, Plasmids, and Cloning

All primers and gene sequences were ordered from IDT DNA Technologies.Primer sequences are included in Table 1; gene sequences are included inSupplementary Materials. All PCRs used Accuprime Pfx (Thermo FisherScientific) with manufacturer's recommended conditions unless otherwisenoted. Standard Gibson assembly techniques were used for all assembliesunless otherwise noted. Shuffle-optimized KlenTaq, Bst LF, and alllibraries for IsoCSR selections were cloned into pLTetO, an in-housedesigned plasmid based on the pASK-IBA37plus vector (IBA GmbH) thatreplaces the pA_(tetO) promoter with a pL_(tetO) promoter (Lutz 1997 andremoves the 6×His tag, multiple cloning site, and Rop gene. Forexpression and purification, polymerase genes were cloned into pATetO6×His, a similar modification of pASK-IBA37plus that retains theN-terminal 6×His tag and a similar pA_(tetO) promoter with a singlepoint mutation to make it unidirectional, but again removes the multiplecloning site and Rop gene, making it high copy. Assembled plasmids weretransformed into electrocompetent Top10 (Thermo Fisher Scientific) orBL21 (New England Biolabs) E. coli strains as described and cultured in2×YT media (Thermo Fisher Scientific) supplemented with 100 μg/mlAmpicillin to maintain plasmid at 37° C. unless otherwise indicated.

(2) Sequences and Analysis

Protein sequences were obtained from the sources indicated in the text.All known Taq mutations (Chen 2014) were mapped onto Taq for comparativeanalysis with the selected libraries. Protein sequences for Bst LF,Klentaq, v5.9, and v7.16 are included in Supplementary Materials. Sangersequencing was utilized for all sequencing. For sequencing after rounds3, 5, and 7, primers JNM245 and JNM259 were used (see Table 1). Forverification after cloning into pATetO for expression, primers JNM135and JNM141 were used, as well as either JNM101 for Bst LF-basedsequences or JNM258 for Klentaq-based sequences (see Table 1). Allsequence assembly and analysis utilized Geneious version 7.1 created byBiomatters. Alignments were performed with 65% identity cost matrix, gapopen penalty 12, and gap extend penalty 3.

(3) Shuffled Library Preparation

Bst LF and Klentaq protein sequences were (Kiefer 1997; Korolev 1995)codon optimized for expression in E. coli using the IDT codonoptimization tool. The resultant DNA sequences were then copied intoShuffle Optimizer, an in-house developed open-source python program thatoptimizes one sequence for DNA shuffling in reference to another(Milligan 2017), FIG. 7). Klentaq was optimized in reference to Bst LF.These DNA sequences were ordered as gBlocks (Integrated DNATechnologies). These sequences were amplified using primers JNM219 andJNM220 (Table S1), such that ˜150 bp of homology to the backbone isadded to each side of the gene for optimal shuffling. Fragments were runon an 0.8% agarose gel, cut, and purified using a Wizard SV Gel and PCRCleanup System (Promega). 5 μg of equimolar concentrations of BstLF andKlentaq were brought up to a volume of 130 μL with deionized water,placed in Covaris microAFA tubes (part number 520045) and added to aCovaris S2 ultrasonicator. The ultrasonicator was run followingmanufacturer's recommendations for DNA shearing to get 150 bp fragmentpeaks. Approximately 500 ng of each fragmented library was included in aprimerless PCR reassembly reaction using Platinum Taq HiFi (Invitrogen)containing 1× Platinum Taq HiFi buffer, 0.2 μM ea. dNTP, and 2 mMadditional magnesium sulfate up to a final volume of 100 μL. Thereactions were incubated on a thermal cycler at 95° C. for 2 min, thensubjected to 35 cycles of the following series of incubations: 95° C.for 30 sec, 65° C. for 90 sec, 62° C. for 90 sec, 59° C. for 90 sec, 53°C. for 90 sec, 50° C. for 90 sec, 47° C. for 90 sec, 44° C. for 90 sec,41° C. for 90 sec, and 68° C. 125 sec. The assembly reactions werepurified using a standard PCR cleanup kit. 50 ng of DNA from eachlibrary assembly were amplified in a recovery PCR reaction with primersJNM221 and JNM222 (Table 1) using AccuPrime Pfx (Thermo FisherScientific). Fragments were run on an 0.8% agarose gel, cut, andpurified using standard techniques. After Gibson assembly andtransformation, serial dilutions of transformed cells were plated tocalculate library efficiency. 20 colonies from each library werecultured, miniprepped, and sequenced.

(4) IsoCSR Mock Selections

The IsoCSR selection was initially optimized using wild-type Bst LF andBstXX, an inactive variant with 5 stop codons and an EcoRI digestionsite. BL21 cells carrying pLtetO plasmids encoding either polymerasewere cultured with increasing ratios of inactive BstXX:Bst LF andinduced with a final concentration of 200 ng/ml Anhydrotetracycline(ATc, Sigma) for 4 hr at 37° C. for protein expression. ˜1.5×10¹⁰ cellswere washed, then resuspended in 150 μL of an aqueous mixture consistingof 1× NEBuffer2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH7.9), 0.4 mM dNTPs, 15 μg BSA, 30 μg lysozyme (Sigma), 1 mM additionaldTTP, 18 U dUTPase (ProSpec), and 75 U Nb.BsmI (Nicking endonuclease,New England Biolabs). The mixtures were then added to 600 μL of oil mix(73% Tegosoft DEC, 7% AbilWE09 (Evonik), and 20% mineral oil(Sigma-Aldrich)) and emulsified using a TissueLyser LT (Qiagen) set to35 Hz for 4 min. Emulsions were distributed equally into PCR tubes andincubated at 37° C. for 20 min (lysis), 65° C. for 3 hr (nicking andRCA) and 80° C. for 20 (to inactivate Bst LF), then cooled to 4° C.Emulsions were broken by spinning the reaction at 10,000×g for 5 min at4° C., removing the top oil phase, adding 150 μL of H₂O and 300 μLchloroform, mixing via pipette, and finally phase separating in a phaselock tube (5Prime). Pooled DNA was purified by ethanol precipitation andresuspended in 75 μL 1×HF Buffer (New England Biolabs). 5 μL of purifiedproduct was digested in 50 μL reactions containing 1×HF Buffer and 40 UDpnI (New England Biolabs) for 12 hr to remove unamplified plasmid DNA.DNA was recovered with 2 rounds of PCR using Phusion polymerase (NewEngland Biolabs) (20 cycles each) with primers JNM182 and 183 for thefirst PCR and JNM102 and 183 for the second. Products were purifiedusing PCR purification kits (Zymo Research) after each PCR reaction.Products were digested in 50 p L reactions containing 1× CutSmart Buffer(50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate,100 μg/ml BSA, pH 7.9) and 10 U EcoRI-HF (New England Biolabs) for 3 hrat 37° C. to distinguish Bst LF from BstXX and analyzed with gelelectrophoresis.

(5) IsoCSR Selection of Thermostable, Strand-displacing Polymerases

Libraries were cloned using Gibson assembly with 2 to 3-fold ratios oflibrary insert to plasmid using 500-1,000 ng pLTetO plasmid backbone,then column purified using PCR purification kits (Zymo Research) andelectroporated into BL21 cells. Typical transformations yielded 10⁶-10⁸cfu. Overnight cultures were diluted 1:20 into fresh media, grown for 1hr at 37° C., induced with a final concentration of 200 ng/ml ATc, andfurther cultured for 4 hr. ˜1.5×10¹⁰ cells were washed with 75 μL 1×PCRbuffer (50 mM KCl, 10 mM Tris HCl pH 8.3, 1.5 mM MgCl₂), and resuspendedin 150 μL of an aqueous mixture containing 1×PCR buffer, 0.4 mM dNTPs,15 μg BSA, and 1, 5, or 10 forward and reverse primers at aconcentration of 0.5 μM each as described in Table 1, JNM264-283. Themixtures were then added to 600 p L of oil mix (73% Tegosoft DEC, 7%AbilWE09 (Evonik), and 20% mineral oil (Sigma-Aldrich)) and emulsifiedusing a TissueLyser LT (Qiagen) set to 42 Hz for 4 min. Emulsions weredistributed equally into PCR tubes and incubated at 95° C. for 5 min(lysis and template denaturation), 65° C. for 3 hr (RCA) and 80° C. for20 min, then cooled to 4° C. Emulsion breaking, DNA purification, andDpnI digests were identical to mock selections. Products were recoveredusing 2-3 rounds of PCR amplification, 20-30 cycles each. Initialrecovery PCRs used primers JNM219 and JNM220 and 1×HF buffer withAccuprime Pfx (Thermo Fisher Scientific), while subsequent PCRs usedJNM221 and 222 with standard Accuprime conditions. PCRs were gel orcolumn purified using PCR purification kits (Zymo Research). Productswere cloned as before and electroporated into Top10 (Invitrogen) cellsfor sequencing or BL21 (New England Biolabs) cells for furtherselections as needed.

(6) Exonuclease III Parasite Removal

PCR library recovery in later rounds of IsoCSR was difficult, asreaction products were overrun with parasites, or small off-targetamplicons. Where indicated, the following procedure was used to removeparasites. After the recovery PCR, products were purified using PCRpurification kits (Zymo Research) and eluted in 6 μL H₂O. 600 ng PCRproduct was added to a 60 μL reaction containing 1×PCR buffer (50 mMKCl, 10 mM Tris HCl pH 8.3, 1.5 mM MgCl₂) and 60 U ExoIII (New EnglandBiolabs). Reactions were assembled on ice, then incubated at 25° C. for8.5 min. ExoIII digests DNA in the 3′ to 5′ direction at ˜100 nt/min at25° C. from both ends of double-stranded DNA, so this incubation isexpected to remove products smaller than 1,700 bp, but can be adjustedas needed. Immediately following incubation, reactions were mixed with30 μL of 1×PCR buffer containing 0.4 mM dNTPs and 3 U Taq Polymerase(New England Biolabs) and incubated at 68° C. for 10 min, which heatinactivates the ExoIII enzyme and enables overlap extension ofincompletely digested products.

(7) Polymerase Purification

Wild-type Bst LF and Klentaq as well as individual variants isolatedfrom selection were cloned into pATetO 6×His (see Cloning above).Products were amplified using primers JNM316 and JNM309 for 5′ and 3′Bst LF ends and primers JNM317 and JNM310 for Klentaq 5′ and 3′ ends,respectively. Plasmids were transformed into BL21 cells. Single colonieswere inoculated into 5 mL Superior Broth (Athena Enzyme Systems)supplemented with 100 μg/ml Ampicillin and grown overnight at 30-37° C.Cultures were diluted 1:200 into 250 mL to 1 L fresh media and culturedto OD₆₀₀ 0.5-1, then induced with a final concentration of 200 ng/mL ATcand further cultured for 3-7 hr for expression. Cells were harvested(4,000×g, 15 min, 4° C.), frozen in liquid nitrogen, and stored at −80°C. Cells were resuspended in 20-40 mL Lysis Buffer (20 mM Tris pH 7.4,300 mM NaCl, 0.1% Tween-20 (Thermo Fisher Scientific), 10 mM Imidazole)supplemented with EDTA-free Protease Inhibitor Tablets (Thermo FisherScientific) and 0.5 mg/mL Lysozyme, and mixed end-over-end for 30 min at4° C. Cells were further lysed using sonication. Supernatants werecleared (40,000×g, 30 min, 4° C.), heated for 65° C. for 20 min withshaking (400 rpm), and cleared again (20,000×g, 20 min, 4° C.).Polymerases were purified by metal ion chromatography. Briefly, lysateswere added to 1 mL pre-equilibrated HisPur Ni-NTA resin and incubatedfor 30 min at 4° C. with end-over-end mixing for batch binding. Thesewere applied to gravity columns and allowed to drain, then washed with3×10 mL Wash Buffer (Lysis Buffer with 40 mM Imidazole) and eluted with4×1 mL Elution Buffer (Lysis Buffer with 250 mM Imidazole).

For initial qLAMP screening, elutions were pooled, dialyzed into StorageBuffer (10 mM Tris pH 7.4, 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5%Tween-20, 0.5% Triton-X100, and 50% Glycerol), and stored at −20° C. Forcharacterization of thermostability and RCA assays, elutions (Bst LF,Klentaq, v5.9) were further purified and instead dialyzed into Buffer A(20 mM Tris, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, and 0.1% Tween-20), thendiluted with 14 mL Buffer A1 (Buffer A without Tween-20). Elutate wasapplied to a gravity column with 1 mL Type I Heparin agarose resin(Sigma) pre-equilibrated with 10 mL Buffer A1, then washed 2×10 mL withBuffer A1. Proteins were eluted on a 0.15 M to 0.8 M NaCl gradient, withpolymerases typically eluting at 470-575 mM NaCl. Elutions were pooledand dialyzed into storage buffer and stored at −20° C. For Nickelpurifications, protein purity was 50-90% as indicated by SDS-PAGEelectrophoresis, typically ˜80%. For Nickel and Heparin purifications,proteins were ≥99% pure. For all assays, protein concentrations wereequilibrated to commercial Bst LF (New England Biolabs) using SDS-PAGEdensitometry. It was chosen to normalize to concentration rather thanactivity in order to accurately compare the functionality of thevariants to their wild-type ancestor polymerases, Bst LF and Klentaq.

(8) qLAMP Screening

LAMP reactions contained 1× Thermopol buffer (20 mM Tris-HCl, 10 mM(NH₄)₂SO₄, 10 mM KCl, 2 mM MgSO₄, 0.1% Triton X-100, pH 8.8, New EnglandBiolabs), an additional 2 mM MgSO₄ (4 mM final concentration), 0.4 mMdNTPs, 20 μg template (GAPDH, Table 2), 1× primer mix (FIP=1.6 uM,BIP=1.6 uM, LR=0.8 uM, F3=0.4 uM, B3=0.4 uM, Table 2), 1M Betaine, 1×EvaGreen fluorescent DNA intercalating dye (Biotium) to monitoramplification, and 2.5 ul polymerase in a total reaction volume of 25μL. Reactions used 5 primers instead of the typical 4 or 6 as inprevious studies (Bhadra 2015). Fully assembled reactions were heatdenatured at times and temperatures indicated in the text prior to LAMPwith or without polymerase included as noted. For SD Pol LAMP tests, SDPolymerase Hotstart was purchased (Bioron), and reactions were assembledaccording to manufacturer's recommendations (1×SD Reaction Buffer, 3.5mM MgCl₂, 15-50 U SD Pol) with the addition of primers, template, andfluorescent dye as mentioned above; these reactions were heated for 2min at 92° C. to activate hotstart before assaying.

Reactions were monitored using a LightCycler 96 quantitative PCR machine(Roche) by incubating at 68° C. and taking FAM fluorescence measurementsevery 4 min, followed by a post-amplification melt curve analysis todetermine product specificity. Curves were analysed using accompanyingsoftware with absolute quantitation and Tm calling analyses. Thus, Cqvalues (where indicated) represent crossing a fluorescent thresholdvalue determined by the software and correspond to a time point(multiply by 4 min) rather than a cycle number as in qPCR.

(9) Thermostability Assays

Kinetic activity assays were performed according to manufacturer'sinstructions using the EvaEZ fluorometric polymerase activity assay kit(Biotium). 10 μL reactions consisting of 1×PCR buffer (50 mM KCl, 10 mMTris HCl pH 8.3, 1.5 mM MgCl₂) and 0.1 μL v5.9 (Nickel and Heparinpurified) were incubated at 85, 89.5, or 92.5° C. for 0, 1, 2, 5, or 10min, then immediately cooled on ice. Reactions were mixed with 10 μL 2×Polymerase Activity Mix and monitored on a Light Cycler 96 with readingsevery 1 min. Initial slopes, indicating reaction rates, were measured.Activity of samples was normalized to the no heating control andexpressed as a percentage.

(10) Rolling Circle Amplification Assays

RCA reactions contained 1× Thermopol buffer (20 mM Tris-HCl, 10 mM(NH4)₂SO₄, 10 mM KCl, 2 mM MgSO₄, 0.1% Triton X-100, pH 8.8, New EnglandBiolabs), 0.4 mM dNTPs, 100 ng pATetO plasmid template (excepting notemplate controls), 1× EvaGreen fluorescent DNA intercalating dye(Biotium) to monitor amplification, and 2.5 ul polymerase (Nickel andHeparin purified) in a total reaction volume of 25 μL. Where indicated,reactions also included 0.5 μM each of 20 primers, 10 forward and 10reverse, for exponential amplification (JNM264-283, see Table 1). Forreactions containing nicked template, 2.5 μg of plasmid pATetO wasnicked in a 50 μL digestion reaction containing 1×NEBuffer 3.1 (100 mMNaCl, 50 mM Tris-HCl, 10 mM MgCl₂, 100 μg/ml BSA, pH 7.9) and 20 UNb.BsmI (New England Biolabs) by incubation at 65° C. for 1.5 hrfollowed by 80° C. for 20 min to heat-kill the nickase. 2 μL of thisreaction or 50 ng/μL non-nicked pAtetO in 1×NEBuffer 3.1 were added toreactions containing template in order to maintain consistency.

Reactions were monitored similarly to qLAMP, using a LightCycler 96quantitative PCR machine (Roche) by incubating at 68° C. and taking FAMfluorescence measurements every 4 min, followed by a post-amplificationmelt curve analysis to determine product specificity. Curves wereanalysed using accompanying software with Tm calling analysis. UnlikeLAMP, RCA reactions do not produce qPCR-like curves that are easilyinterpretable by the Light Cycler 96 software. Thus, all RCA datapresented is unprocessed fluorescence data, and therefore does not haveCq values.

(11) Structural Analysis of Polymerases

Protein structure files (PDB) were obtained from RCSB (Berman 2000). Thestructures used in this study were 113s for Bst LF and 3 ktq for Klentaq(Kiefer 1998; Li 1998). Alignments and other structural figures wereprepared using pymol (The PyMOL Molecular Graphics System, Version 1.8Schrödinger, LLC).

b) Results

(1) Isothermal Selection Schemes for DNA Polymerases

While there are a number of schemes for the self-selection ofthermophilic DNA polymerases for PCR applications, there have previouslybeen few ways to select functional polymerases that work at moremoderate temperatures and for end uses other than PCR. A novel selectionstrategy termed term isothermal compartmentalized self-replication(IsoCSR, FIG. 1) has been developed. In this scheme, libraries ofpolymerase variants are expressed in cells, and cells are ensconcedwithin individual compartments in water-in-oil emulsions. However, genesthat produce functional polymerases are not amplified by PCR, as is thecase with techniques such as compartmentalized self-replication (CSR)that rely on thermal cycling to disrupt cells, but rather via rollingcircle amplification (RCA) and enzymatic lysis of cells. Thus, thescheme is able to accommodate mesothermophilic (functional up to 70° C.)strand-displacing polymerases, such as the widely used Bst polymerase.Others have recently used an isothermal variant of compartmentalizedself-replication to evolve phi29 polymerase, but this method relied onrepeated freeze-thaw cycles to lyse emulsified cells and would not besuitable for a more thermostable polymerase (Povilaitis 2016).

IsoCSR begins by transforming E. coli with an expression plasmidcarrying a library of polymerase genes cloned downstream of an induciblepromoter (FIG. 1a ). Polymerase expression is initiated withanhydrotetracycline (ATc), after which the cells are harvested andresuspended in an aqueous mixture containing buffer, dNTPs, ahigh-temperature nicking endonuclease (Nb.BsmI, New England Biolabs),and lysozyme. This mixture is emulsified using established methods(Ellefson 016; Kiefer 2014). Cells are lysed within the emulsioncompartment enzymatically by lysozyme during an initial 37° C.incubation, releasing polymerase and plasmid. Raising the temperature to65° C. activates the thermostable nickase, which nicks the plasmid toenable linear RCA via strand-displacing polymerization. After thereaction, emulsions are broken and genes encoding variants that havebeen successful at RCA can be further enriched by PCR. Multiple cyclesof emulsion, expression, RCA, extraction, and PCR are anticipated toyield polymerases that are highly functional under isothermal, ratherthan thermal cycling, regimes.

IsoCSR was attempted (Kiefer 1997) with Bst LF, a mesothermophilicenzyme that is especially interesting because of its strand-displacementproperties, facilitating its use in powerful isothermal amplificationschemes such as rolling circle amplification (RCA) (Zhang 2001),helicase-displacement amplification (HDA) (An 2005), and loop-mediatedisothermal amplification (LAMP) (Notomi 2000). RCA was focused on forself-amplification as it can most readily reproduce full-length genes.Enzymatic lysis with lysozyme and template amplification via themesothermophilic nicking endonuclease Nb.BsmI was designed and verified,which generates an RCA initiation site on the expression plasmid.In-emulsion cell lysis, nicking, and RCA were all verifiedindependently, then combined for further optimization (see SelectionOptimization).

Once the selection protocol was fully optimized, a mock selectionexperiment was carried out to investigate whether functional polymerasescould be enriched relative to a nonfunctional mutant. The non-functionalvariant BstXX was created by inserting 6 stop codons and a unique EcoRIcut site upstream of the active site of Bst LF. IsoCSR was thenperformed with ratios of 1:10, 1:100, and 1:1000 Bst LF:BstXX cells. PCRrecovery products were digested with EcoRI to differentiate between BstLF and BstXX. The mock selection demonstrated that it can be possible torecover wild-type Bst LF even in the presence of 10³ excess inactivemutant Bst (FIG. 2), a ratio that is similar to that observed for someother polymerase selections (Chen 2016) and significantly improves uponreports with phi29 polymerase (Povilaitis 2016), but was less efficientthan CSR, which was reported to recover active variants in the presenceof a 10⁶ excess of an inactive mutant (Ghadessy 2001). This could be dueto the linear nature of rolling circle amplification and to therequirement that multiple enzymes (polymerase, nickase, lysozyme)function within the emulsion.

To improve the selection coefficient and recovery efficiency, a modifiedIsoCSR selection was contemplated in which nickase and lysozyme werereplaced with thermal denaturation, similar to the original CSRprotocol, yet still requiring strand displacement activity for RCA.So-called thermostable IsoCSR, which includes a pre-RCA heatdenaturation step for cellular lysis and template denaturation, alsoallowed primer-initiated hyperbranched RCA, which has exponential ratherthan linear reaction kinetics. However, because this embodimentnecessitated thermostability in addition to strand displacementactivity, neither wild-type Bst LF nor other strand-displacingpolymerases could effectively carry out the cycle. A thermostableversion of Bst was therefore created that could in turn be used toinstantiate thermostable isoCSR.

(2) Library Generation

DNA shuffling has proven useful as a means of generating diverse proteinlibraries, especially for selecting polymerases with novel phenotypes(Baar 2011; Stemmer 1994; Crameri 1998). A library for thermostableisoCSR was created by shuffling two family A polymerases: thestrand-displacing DNA polymerase I from Bacillus (Geobacillus)stearothermophilus (Bst) and the thermostable Thermus Aquaticus (Taq).The large (Klenow) fragments of these polymerases, Bst LF and Klentaq,are truncated versions that lack the N-terminal 5′-3′ exonucleasedomain, and thus promote strand displacement activity, although todifferent degrees (Kiefer 1998; Lawyer 1993). Bst LF and its engineeredhomologs are the polymerases of choice for high temperature isothermalamplification reactions due to their high strand displacement activity(Mori 2013; Notomi 2000; Lizardi 1998; Zhang 2001), but are unstable attemperatures above 70° C. In contrast, Klentaq has much weaker stranddisplacement activity but is more thermostable than full-length Taq,with a half-life of 21 min at 97.5° C. (Lawyer 1993). Through molecularbreeding of these related-but-phenotypically-different enzymes, achimeric variant was selected that would combine both activities.

DNA shuffling requires high levels of homology between molecules forsuccess (Baar 2011). However, Bst LF and Klentaq were only 53% identicalat the DNA level, which proved to be insufficient for shuffling methods.Increasing homology at the DNA level leads to increased shuffled librarydiversity, and has previously been accomplished by computer programsthat optimized codons to maximize stretches of homology between DNAmolecules without changing the resulting protein (Moore 2000; Moore2002; Moore 2001). The lack of ready availability of these programs ledus to develop the development of a Python script called ShuffleOptimizer (Milligan 2017), now available as open-source code for publicuse (FIG. 7). Shuffle Optimizer generated an optimized Klentaq DNAsequence that was nearly 70% identical to Bst LF DNA, a significantimprovement over the original sequence identity (53%).

Optimized sequences were then shuffled. Ultrasonication was used togenerate 150 bp DNA fragments of Bst LF and optimized Klentaq, and thesefragments were then mixed in equal ratios and reassembled using overlapextension PCR (see also Methods). Shuffling of the optimized sequencesresulted in a library of ca. 5×10⁷ variants, of which 14 out of 20unselected variants were chimeric, with an average of 1.8 crossovers perchimeric variant (FIG. 8). A side-by-side attempt to shuffle sequencesnot optimized by Shuffle Optimizer resulted in 0 observed crossovers in20 variants. Notably, few crossovers were observed near the 5′ ends ofthe genes, likely due to high sequence divergence in this region evenafter optimization. Nevertheless, Shuffle Optimizer enabled thesuccessful creation of a shuffled protein library from Bst LF andKlentaq sequences where it had previously failed, and should be usefulfor other researchers interested in shuffling or recombining distantlyrelated sequences.

(3) Selection of Thermostable, Strand-Displacing Polymerases

Thermostable polymerases capable of isothermal amplification wereselected by carrying out hyperbranched rolling circle amplification(hbRCA) in emulsio. The use of hbRCA greatly simplified the selection byeliminating the need for nicking endonuclease and lysozyme, as thesesteps were replaced with a pre-RCA denaturation step of 95° C. at 5 minthat both lysed cells and denatured the plasmid for primer binding (FIG.1b ). In addition, hbRCA likely increased the selection coefficient foractive variants because it involves exponential reaction kinetics (Zhao2015; Dean 2001; Lizard 1998; Zhang 2001).

This selection can yield thermophilic enzymes, as amplification isdependent on the polymerase surviving the initial denaturation step. Thepolymerase must also retain or acquire strand displacement activity inorder to perform hbRCA. Interestingly, while Klentaq had sufficientstrand displacement activity to carry out RCA on nicked templates, itinitially failed to amplify plasmid DNA in a hbRCA reaction (see FIG. 5bversus 5c). On the other hand, Bst LF has strand-displacing activitysufficient for hbRCA, but lacks thermostability. Thus, neither wild-typepolymerase used to generate the library—indeed, no polymeraseknown—possessed the combination of activities necessary to survive theselection, a strong argument for the selection of a chimera.

7 rounds of IsoCSR were turned to evolve novel thermostable,strand-displacing variants. 20 primers were utilized in the emulsionhbRCA reaction, with 10 forward and 10 reverse primers equally spacedaround the non-library portion of the expression plasmid at aconcentration of 0.5 μM each. The stringency of the selection wasincreased over successive rounds by scaling down the number of primersincluded, as the number of primers positively correlated withreplication efficiency (Dean 2001). This scaling methodology requiredoptimization to ensure successful recovery of the library, as overlyhigh selection pressures resulted in no product recovery frompost-emulsion PCR. Indeed, post-emulsion library recovery in laterrounds with only two hbRCA primers was challenging, as reactions weredominated by parasites, small off-target PCR products that werepreferentially amplified over the larger (˜1800 bp) polymerase geneproduct. To circumvent these difficulties a novel parasite removalstrategy was developed that relied on exonuclease III (ExoIII, NewEngland Biolabs), a synchronous 3′ to 5′ DNA exonuclease withwell-studied reaction kinetics (Hoheisel 1993; Roychoudhury 1977; James1984). By lowering the digestion reaction temperature to 25° C., whereExoIII cuts at a rate of ˜100 bp, it was found that 8 minutes oftreatment would completely digest products less than 1700 bp in length,while leaving overhangs on larger products. The remaining hemiduplexesserved as substrates for primer extension by Taq polymerase at 68° C. (atemperature that also denatured ExoIII). The combined ExoIII digestionand Taq polymerase extension resulted in the recovery of only thedesired large product from later rounds of selection (FIG. 9), and ledto successful library recovery from IsoCSR in Rounds 5, 6, and 7.

In addition to seeing how the selection could respond to increasedstringency (limiting the primers required for successful amplification),its progress was assessed by sequencing 12-20 individual variants fromRounds 3, 5, and 7. Most polymerase variants were chimeras composed ofBst LF and Klentaq. In later rounds, these insertions appeared toconverge. In Round 5, over half (7/13) of the observed insertionsoverlapped at the C terminus of the protein (Klentaq residues530-535/Bst LF residues 570-575); this had increased to 75% of thechimeric population by Round 7.

Increasing numbers of non-synonymous mutations were also observed oversuccessive rounds, presumably due to replication errors that aroseduring self-replication and then became fixed. Several concentratedclusters of mutations occurred in Round 7 at or near previouslyidentified mutations in Taq that led to a relaxation of substratespecificity (Ghadessy 2001; Chen 2014; Laos 2013; Leconte 2010; Suzuki1996; Vichier-Guerre 2006). Relaxed substrate specificity is oftensynonymous with decreased fidelity (Chen 2014), and it appeared that thelibrary was becoming enriched for error-prone polymerases with increasedreplication rates, as has been seen in previous selections (Suzuki 1996;Patel 2001; Aryan 2010).

(4) Variant Purification and Screening

Since it seemed from sequencing results as though there was no consensussequence for thermostable, strand-displacing polymerases, for initialscreening, twelve variants from Round 7 and six variants from Round 5were cloned into a protein expression vector with an N-terminal 6×His.Of the 18 polymerases cloned, 5 variants could not be expressed andpurified in sufficient quantities for screening; these latter largelyconsisted of proteins that had a Klentaq backbone with a C-terminal Bstinsertion.

The 13 purified variants were screened for thermostability and stranddisplacement activity using loop-mediated isothermal amplification(LAMP) with a well-known GAPDH template as an assay, as this combinationcan readily yield interpretable qPCR-like exponential amplificationcurves. qLAMP Reactions were assembled and monitored on a LightCycler 96qPCR machine (Roche) as previously described (Jiang 2015; Zhang 2001),except that EvaGreen intercalating fluorescent dye (Biotium) was usedrather than oligonucleotide probes (see also Methods). Ampliconhomogeneity was monitored via post-reaction melt curves (Njiru 2008). Asecondary screen was also carried out using RCA with a nicked plasmidtemplate at 68° C.; this assay requires less robust strand displacementcapability.

Initial screening identified two highly functional variants capable ofLAMP and RCA, v5.9 from Round 5 and v7.16 from Round 7 (FIG. 3a ). Cqvalues, which represent the time at which fluorescence exceeds adetermined threshold value (see Methods), indicated that v5.9 was 24.7min slower (Cq=16.6) than purified wild-type Bst LF (Cq=10.5), whilev7.16 was 7.3 min faster (Cq=8.6), all with similar melt peaks. v5.9 andv7.16 were compared with SD Pol (Bioron), a commercially available Taqmutant reportedly capable of LAMP (Ignatov 2014); however, it was foundthat this enzyme was unable to amplify products via LAMP underrecommended conditions, though it was capable of RCA from a nickedplasmid template. Additional variants (v7.5) that were not LAMP capablecould also carry out RCA.

(5) Sequences of Functional Polymerase Variants

Sequencing data showed that variant 5.9 had a Klentaq backbone with asmall Bst insertion and two point mutations, while variant 7.16consisted of Bst LF with no insertions and four point mutations. Theinsertion in v5.9 is located near other insertions observed in Round 5,but did not contain an insertion in the C-terminal region where mostinsertions converged. Similar polymerases with C-terminal insertions mayhave been lost in the initial screen, as chimeras containing C-terminalBst LF insertions could not be purified in sufficient quantities fortesting, as described above. V7.16, had several mutations (Q249R, N416S,E453G) occurring at or near known Taq mutations related to decreasedfidelity, similar to other sequenced variants from Round 7. Two of thesemutations (N416S, E453G) were within highly-mutated regions in Round 7,while an additional mutation (Q249R) aligned to a Taq mutation involvedin unnatural nucleobase incorporation.

(6) Thermal Stabilities of Evolved Polymerase Variants

One key advantage of a more thermostable strand-displacing polymerase isthe ability to carry out isothermal amplification reactions that rely onstrand separation, such as LAMP, with an initial strand denaturationstep or at higher temperatures in general. To that end, ‘one pot’reactions were set up with V5.9 and v7.16 in which the entire reactionwas pre-heated for 1 min at 85° C., 89.5° C., or 95° C. prior tocarrying out the remainder of the qLAMP amplification reaction.Remarkably, variant 5.9 successfully performed LAMP after heating at allthe tested temperatures, while neither Bst LF nor v7.16 were able toamplify after any of the thermal challenges (FIG. 3b ). Variant 5.9performed just as well after heating at 85° C. or 89.5° C. (FIG. 3b ) asit did in assays without pre-heating (compare with FIG. 3a ). Increasingheating time from 1 min to 2 min did not further affect performance(FIG. 12). These results show that v5.9 can be used in much morestreamlined LAMP assays in which an initial thermal denaturationprovides primers with uniform access to templates, including G:C richtemplates, which can lead to orders-of-magnitude improvements in assaysensitivity (Njiru 2008; Sagner 1991; Kong 1993). Because the evolvedpolymerase is thermostable, it can now be directly included during thedenaturation step, circumventing an unwieldy workflow that would requireopening tubes between heat steps, something that is particularly onerouswith multiple samples in parallel. This advance also affords for thepossibility of including thermal denaturation in lab on a chipdiagnostic devices that use isothermal nucleic acid amplificationmethods.

In order to further characterize the thermal tolerance of variant 5.9,activity assays were performed similar to radioactivity-based assayspreviously described (Ghadessy 2001; Lawyer 1993; Sagner 1991), exceptthat the EvaEZ fluorometric polymerase activity assay kit (Biotium) wasused to monitor reactions in real-time. Activity was determined bymeasuring initial reaction rates after heating at 85° C., 89.5° C., or92.5° C. for 1-10 min and normalizing these to activity without heating(FIG. 4). V5.9 retained full activity after heating at 85° C. for up to10 min. At 89.5° C., the enzyme had a half-life of approximately 3.75min, while at 92.5° C., the half-life was ˜1.3 min. This represents asignificant decrease in thermal tolerance from the variant's parentenzyme, Klentaq, which has a half-life of ˜21 min at 97.5° C. and shares97.5% protein sequence identity with v5.9.

(7) Assay performance of Variant 5.9

The combination of thermostability and strand displacementcharacteristics found in v5.9 can prove useful in other reactions aswell. v5.9 was tested against its parent enzymes in high temperaturerolling circle amplification reactions, including both linear (singleinitiation) and exponential (multiple primer, hyperbranched) RCA(Lizardi 1998).

A nicked plasmid was used to monitor linear RCA and strand displacementactivity at high temperatures similar to other strand displacementassays (Kong 1993), and melt curve analyses were carried out to ensurethat only specific products were being amplified. Reactions weremonitored via EvaGreen incorporation on a LightCycler 96 qPCR machine(Roche). The plasmid pTetA-6×His was used as a template, and was nickedat a single site with Nb.BsmI (New England Biolabs). Bst LF, Klentaq,and v5.9 were all able to amplify DNA using linear RCA from a nickedplasmid template to varying degrees (FIG. 5a ). V5.9 produced thelargest signal and fastest rate, while Bst LF reached an early plateau,despite initially producing signal faster than v5.9. Klentaq, a weakstrand displacer, produced very little signal, yet had melt curve peakssimilar to Bst LF and v5.9, indicating at least some amplification (FIG.13a ). No template negative controls generated no signal, as expected.

When primers were included with the nicked template (the same 20primers—10 forward and 10 reverse—used in the selections) and sampleswere pre-heated at 95° C. for 1 min to allow primer binding, all threepolymerases were capable of exponential hbRCA amplification (FIG. 5b ).Variant 5.9 and Bst LF produced nearly identical curves, while Klentaqproduced lower signal due to its weak strand displacement activity. Notemplate controls produced small amounts of non-specific signal asindicated by melt curve analysis, likely from the extension of primerdimers, a common issue in multiply primed hbRCA (FIG. 13b ).

Supercoiled template hbRCA reactions are similar to randomly primed RCAreactions, which typically use low temperature Phi29 polymerase toamplify genomic and plasmid DNA (Nelson 2002; Reagin 2003). In addition,hyperbranched RCA reactions with non-nicked, supercoiled plasmidtemplates are similar to the reaction mechanism that was actually usedin the IsoCSR selections. v5.9 proved to be the only polymerase capableof exponential amplification from the supercoiled plasmid templates(FIG. 5c ). The curve produced by v5.9 appears to be biphasic, producinga small amount of amplification and plateauing before reachingexponential amplification. While Bst LF initially produced someamplification, the curve appears to be linear and plateaus prior toreaching an exponential phase. Klentaq is unable to produce any product,as confirmed by melt analysis (FIG. 13c ). Note that the no templatecontrols for this experiment are identical to FIG. 5b ; this is becauseall experiments were run simultaneously, and the no template controlsfor exponential hbRCA are the same regardless of whether the template isnicked or not in positive reactions.

Overall, v5.9 has superior strand displacement activity to Bst LF inboth linear and hyperbranched RCA, and has acquired the unique abilityto exponentially amplify supercoiled plasmid DNA via hyperbranched RCA(an activity that is not present in either of the polymerases used togenerate the shuffled library). The only other polymerase known to becapable of exponential RCA from supercoiled templates is Φ29, largelyconsidered the most processive strand displacement polymerase known(Blanco 1989; Nelson 2002; Reagin 2003).

(8) Correlating Structure and Function of v5.9

Sanger sequencing had revealed that variant 5.9 is a chimera consistingmostly of Klentaq, with a 14 aa insertion of Bst LF (Bst LF residues 305to 318) resulting in 11 mutations (D283E, P284G, P286L, D287K, L288V,I289V, H290R, R292D, G294K, R295K, L296V) as well as 2 silent mutationsand 2 non-synonymous mutations, E322G and L484S (FIG. 6b ). The Bst LFinsertion was unique, but similar insertions were also found.

In the original crystal structure characterizations of Bst LF andKlentaq (Kiefer 1997; Korolev 1995), it was noted that increased ratiosof certain amino acids (E:D, L:I, and R:K) and increased numbers ofprolines can be indicative of higher thermal tolerance. Thus, it isnotable that the mutations observed in v5.9 shift most of these ratiostoward decreased thermostability when compared to Klentaq: 3 leucineswere lost compared to only one isoleucine, 3 lysines were gainedcompared to a loss of one arginine, and two prolines were lost. Thiscorrelates to the observed loss of thermostability of v5.9 relative toKlentaq.

In order to provide a structural context for the observed functionalproperties of variant 5.9, the structure of v5.9 was modelled incomparison to its ancestors, the large fragment of Bst and Taqpolymerases (Bst LF and Klentaq). When the Bst LF and Klentaq structuresare aligned, the structures are quite homologous, especially in thefinger and thumb subdomains (FIG. 6a ). The crossover region was mappedwhere the Bst LF insertion occurred in v5.9 onto the structures ofKlentaq and Bst LF for comparison. The inserted region was located inthe polymerase domain at the base of the thumb subdomain, just below theI helix (FIG. 6a ). The insertion observed in variant 5.9 differedsignificantly between the two protein structures (FIGS. 6c and 6d ,blue). In Klentaq (FIG. 6c ), the original region is largelyunstructured, with a small alpha helix (denoted as *) near the I helix.In Bst, the homologous region (FIG. 6d ) has a larger alpha helix,essentially extending the length of the I helix. This I helix is anessential structure of the thumb subdomain, forming an antiparallelcoiled-coil structure with the neighboring H helix that is dependent onhydrophobic interactions between leucine residues on the two helices(Kiefer 1997). In addition to extending the I helix, the Bst insertionobserved in v5.9 results in an additional leucine residue at the base ofthe structure (denoted as x), which can show that the inserted sequencecould increase interactions between the I and H helices, stabilizing thethumb subdomain.

In further support of this model, Bst also has a small antiparallel betasheet in this region (FIG. 6d , 3 black arrows) that is not observed inKlentaq, consisting of three interacting beta strands at the base of theI, H, and K helicies of the thumb domain. One beta strand contributingto this structure (FIG. 6d , left arrow) is contained in the insertedregion in v5.9, while the other beta strands consist of identical orsimilar residues in both structures. Given this, the observed insertioncould also enable the formation of a beta sheet at the base of the thumbsubdomain in variant 5.9, which may involve the nearby E322G pointmutation (FIG. 6c ).

Overall, it appears that the Bst insertion observed in v5.9 stabilizesthe structure of the thumb subdomain of the polymerase by extending theI alpha helix and allowing the formation of a beta sheet at thesubdomain's base. This offers a new insight into the structural basis ofstrand displacement activity in polymerases, and with the finding thatmultiple crossovers in this region were observed in the librarypotentially provides a more direct path to engineering stranddisplacement in any polymerase.

(9) Selection Optimization

Designing a polymerase selection for strand displacement poses severalunique challenges compared to typical CSR. First, no thermophilicpolymerase has been shown to be capable of RCA. For this reason, initialoptimization of the parameters and methods necessary for IsoCSR reliedon enzymatic lysis with lysozyme to break open cells and plasmid DNAnicking using the Nb.BsmI restriction endonuclease (New England Biolabs)to initiate rolling circle amplification rather than heating toaccommodate wild-type Bst LF. Enzymatic lysis was optimized bymonitoring emulsified E. coli expressing GFP on an inverted fluorescentmicroscope. Emulsions held at 37° C. for 30 min for lysozyme digestionfollowed by heating at 65° C. for 30 min (simulating the RCA step) werefully lysed in emulsion bubbles, while those held at 4° C. with andwithout lysozyme for 60 min did not lyse (FIG. 14). Lysis was latershortened to 20 min, as this proved to be sufficient.

The emulsified nicking RCA mechanism for IsoCSR was first optimizedwithout cells by including commercial Bst LF (New England Biolabs) andplasmid template in the emulsions, producing a characteristic RCA“smear” when analysed by gel electrophoresis. DNA was purified from therecovered emulsion by ethanol precipitation, as typical DNA columns areinefficient for purifying the large molecular weight products of RCA.Additionally, a DpnI digest (37° C. for 2 hr) was included after DNArecovery from emulsion RCA to reduce background from unamplifiedplasmids. Emulsion RCA product was unaffected by DpnI when analysed bygel electrophoresis, and the subsequent recovery PCR produced a bandcorresponding to the Bst gene product only when polymerase was includedin the initial emulsion RCA.

These conditions were further refined to accommodate E. coli cellsexpressing Bst as a source for both plasmid template and polymerase, ascell lysate inhibited the combined nicking and RCA reaction. This wasaccomplished by increasing emulsion compartment size through reductionof the frequency of emulsification from 42 Hz to 35 Hz, as lowerfrequency emulsions were unstable. These larger emulsions were stable at65° C. for 3 hr, sufficient for RCA in emulsio (FIG. 15). Thisoptimization resulted in successful PCR recovery from emulsion RCAproducts generated from cellular polymerase and template, validating theIsoCSR mechanism. Further optimization benefited from including dUTPaseto remove dUTP, a known Bst inhibitor, and increasing the volume of theaqueous component of the emulsions. In spite of these improvements,nicking IsoCSR was only able to recover wild-type Bst LF in 103 excessinactive mutant Bst (FIG. 2), likely due to the complex mechanismreliant on multiple enzymes to circumvent boiling and linear nature ofRCA initiated from a single nick site on the plasmid template. For thisreason, IsoCSR was adapted to accommodate hyperbranched RCA, enablingexponential amplification (Lizardi 1998; Zhang 2001; Zhao 2015).

c) Discussion

Compartmentalized self-replication (Ghadessy 2001) typically relies onmultiple thermal cycling steps and has been used primarily withthermophilic polymerases to evolve novel functionalities. Thedevelopment of IsoCSR is described herein, a directed evolution methodthat relies on self-replication of less thermostable strand-displacingpolymerases. The Family A polymerases Bst LF and Klentaq were shuffledand the chimeric library was then iteratively challenged for the abilityto carry out strand displacement polymerization following thermalchallenge. Functional strand-displacing polymerases were still recoveredfrom Rounds 5 and 7 (FIG. 3a ) through the introduction of carefullytuned increases in selection pressure (in the form of reduced numbers ofprimers for amplification). The selection was successful in evolvingv5.9, a thermostable polymerase with improved strand displacementcapability. This chimera proved to be useful for carrying out hot startLAMP and a variety of high temperature RCA reactions.

During the development of isoCSR, a variety of experimental hurdles hadto be overcome. In order to bridge the phylogenetic distance betweenFamily A polymerases with desirable phenotypes was developed usingShuffle Optimizer, an open access algorithm that can be used to increaseoverlaps between distantly related DNA sequences for DNA shuffling. Theexonuclease exoIII was used to eliminate small, off-target parasiticproducts that arose during PCR Finally, sequencing data showed that thepolymerase library had become error-prone by Round 7 and in consequence“cheater” polymerases like v7.16 were recovered that survived theselection despite lacking thermostability, likely due to increasedreplication rate at the expense of fidelity. This has been seen in otherCSR selections as well, and mutations that impact fidelity whileretaining valuable new phenotypes can often be repaired throughjudicious reversion to wild-type residues.

IsoCSR is notable for being an emulsion-based selection that can allowlarge libraries to be sieved, but requires only a single thermal orenzymatic lysis step to accommodate both thermostable and mesophilicpolymerases. Recently, Povilaitis and co-workers developed a similarisothermal, emulsion-based directed evolution scheme that relied onwhole genome amplification (WGA) to evolve a phi29 polymerase mutantwith slightly improved thermostability (up to 42° C.) and increasedamplification rate.

However, their selection required pre-emulsion treatment with lysozymeto lyse cells, a step that can lead to cross-contamination of variantsbefore emulsified compartments are produced. Additionally, WGAnecessitates the use of random primers and thus the burden of amplifyingnot only the gene of interest but genomic DNA. Over many cycles, thismay hamper efficient self-amplification and lead to artefact production.In contrast, the IsoCSR selection requires only in emulsio lysozymeincubation or a single heating step to accomplish lysis (FIG. 1, FIG.2), leaving cells intact prior to compartmentalization.

Variant 5.9 is useful for a variety of applications. The enzyme survived1-2 min template denaturation steps at ˜90° C., which were sufficientfor complete template denaturation in LAMP assays. The ability tosubject pre-assembled isothermal amplification reactions to hightemperature incubations is be useful for diagnostic applications, aspre-reaction heating can improve assay sensitivity, reduce amplificationinhibition from crude clinical samples, and serve as a nucleic acidextraction method for the direct detection of viruses and bacteria. Such“one pot” reactions can be especially useful for point of careapplications, where the high temperature step could now be included inlab on a chip devices. Furthermore, variant 5.9 performed similarly orbetter than Bst LF in all the isothermal mechanisms tested; its superiorperformance in both standard linear RCA reactions and atypicalhyperbranched RCA from supercoiled templates can actually make v5.9preferable for many applications.

Variant 5.9 and its shuffled brethren also offered surprising insightsinto the structural and mechanistic properties that underlie the poorlyunderstood process of strand displacement. For instance, it issignificant that a polymerase known for strand displacement activity(Bst LF) is unable to amplify DNA from supercoiled plasmids usinghyperbranched RCA, a mere 14 residue insertion of Bst LF into Klentaqalongside two point mutations enables this, as well as stranddisplacement sufficient for LAMP and relaxed template RCA reactions. Theidentification of this insertion can lay the foundation forunderstanding the structural basis for strand displacement, as itappears that small changes in the secondary structure at the base ofKlentaq's thumb subdomain are sufficient to enable a versatile stranddisplacement phenotype.

IsoCSR can allow the directed evolution of many different polymerasephenotypes including altered nucleotide specificity, resistance toinhibitors, and the utilization of new templates. The initial selectionoptimization with wild-type Bst LF shows that lysozyme-mediated IsoCSRcan be used to optimize multi-enzyme reactions, such as the polymeraseand nickase together for RCA. Such co-optimizations can greatly improvediagnostic applications, whose efficiency can often be stymied bysomething as simple as a dissonance in buffer conditions betweenenzymes.

2. Example 2: Phosphorothioated Primers Lead to Loop-Mediated IsothermalAmplification at Low Temperatures

a) Introduction

In order to lower the operating temperature at which LAMP reactions canbe carried out, a fundamental biophysical principle was exploited: thefact that phosphorothioate residues destabilize helices. Boczkowska andco-workers have carried out thermodynamic studies on the stability ofduplexes formed between phosphorothioate (PS)-modified ssDNA andcomplementary phosphodiester (PO)-modified ssDNA (Boczkowska et al.2002), and reported that the PS modifications substantially reduced themelting temperature of PS-PO dsDNA. This in turn allows more breathingat the termini of dsDNA, and can promote the formation of foldbackhairpins for extension during LAMP. Based on this phenomenon, adifferent amplification method has been developed:phosphorothioated-terminal hairpin formation and self-priming extension(PS-THSP) (Jung 2016), in which the incorporation of phosphorothioate(PS) modifications lead to improved self-folding efficiency of terminalhairpins (LaPlanche 1986).

By incorporating phosphorothioate (PS) modifications into the foldbackprimers used for LAMP a more generic mechanism (PS-LAMP) has beencreated for low temperature amplification. PS-LAMP can operate attemperatures as low as 40° C. with sensitivities that are similar toregular LAMP. The PS-terminated DNA can also display enhanced stabilityagainst degradation by various nucleases that may be present inbiological samples, further enhancing the applicability of PS-LAMP forpoint-of-care (POC) diagnostics.

b) Materials and Methods

(1) Reagent

All chemicals were of analytical grade and were purchased fromSigma-Aldrich (St. Louis, Mo., U.S.A.) unless otherwise indicated. Allenzymes including Bst 2.0 DNA polymerase, ET SSB and RecA were obtainedfrom New England Biolabs (NEB, Ipswich, Mass., USA). Middle Eastrespiratory syndrome (MERS) 1a, 1b and Neuropilin 2 (NRP2) DNA plasmidtemplates were generated as described previously (Jiang et al. 2017;Bhadra et al. 2015). Human genomic DNA was obtained from Promega(Madison, Wis., USA). All oligonucleotides were ordered from IntegratedDNA Technology (IDT, Coralville, Iowa, U.S.A.). Oligonucleotidesequences are summarized in Table 4.

(2) One-Step Strand Displacement (OSD) Probe Preparation

An OSD stock solution was prepared by annealing 10 μM Reporter F with 50μM Reporter Q in 1× Isothermal Buffer (20 mM Tris-HCl, 10 mM (NH₄)₂SO₄,10 mM KCl, 2 mM MgSO₄, 0.1% Triton X-100, pH 8.8). The solution wasincubated at 95° C. for 5 min followed by slow cooling to roomtemperature at a rate of 0.1° C./s. This OSD probe was then kept on icebefore use.

(3) Real-time LAMP with OSD probe

In a typical experiment, reaction mixtures containing templates freshlydiluted in specific copies, 20 pmol each BIP and FIP primers, 5 pmoleach B3 and F3 primers, 10 pmol loop primer, 25 pmol betaine, 10 nmoldNTPs and 36 pmol urea in a total volume of 20 μL of 1× IsothermalBuffer (20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10 mM KCl, 2 mM MgSO₄, 0.1%Triton X-100, pH 8.8) were heated to 95° C. for 5 min. The solutionswere chilled on ice for 2 min, and then 5 μL stock solution containingan OSD reporter (at a final amount of 1.5 pmol Reporter F), 60 U of Bst2.0 DNA polymerase and 0.5 μg of ET SSB (or certain amount of RecA) wasadded to initiate LAMP reactions. Subsequently, 25 μL of the LAMP-OSDsolutions were transferred into a 96-well plate. The reactions wereanalyzed using the LightCycler 96 real-time PCR machine (Roche, Basel,Switzerland) that was set up to incubate the samples for 45/50/60 cyclesof two-step incubations-step 1: incubation at 40/45/60/65° C. for 150sec, step 2: incubation at 40/45/60/65° C. for 30 sec (total incubationtime of 3 min/cycle unless otherwise indicated). The resulting data wasanalyzed using the LightCycler 96 analysis software to generate Cq(quantification cycle) values for each amplification and Cq wasredefined as DT (detection time, min) by multiplying Cq by 3.

c) Results and Discussion

(1) Design of PS-LAMP Reactions

In a typical LAMP reaction (FIG. 1), there are at least four primers:two inner primers (FIP and BIP) and two outer primers (F3 and B3). Theseprimers are specific to six consecutive blocks of target sequences: B3,B2, B1, F1c, F2c and F3c (from the 5′-end of the amplicon). Both theinner and outer primers anneal to the target template and are extendedsimultaneously. The extension of the outer primer with a stranddisplacing polymerase, such as Bst, therefore displaces the innerprimer, which can then fold back on itself to create a dumbbell-shapedamplicon. The inner primers can hybridize to the single-stranded loopsin the foldback structures and initiate another round of stranddisplacement synthesis, forming a concatamer amplicon with aself-priming 3′-hairpin. The ensuing exponential amplification duringcontinuous strand displacement DNA synthesis generates increasinglycomplex, double-stranded concatamer amplicons. The single-stranded loopsin these amplicons can be used to trigger sequence-specific strandexchange reporters, as we have previously described (FIG. 21) (Bhadra etal. 2015; Jiang et al. 2015). These reporters greatly reduce backgroundsignal and provide greater surety in the detection of LAMP amplicons.

The loop structure is key to the amplification mechanism in LAMPreactions. Attempts were made to improve the formation of the foldbackstructure in the extended dsDNA (red box) replication intermediate. Allthe internucleotide linkages in the F1c portion of the FIP primer (20phosphates for MERS 1a and MERS 1b) and in the B1c portion of the BIPprimer (23 and 21 phosphates for MERS 1a and MERS 1b, respectively) weremodified with phosphorothioates (see also FIG. 16). The F2 region in FIPand the B2 region in BIP were excluded from phosphorothioatemodification as this might have reduced priming efficiency. The PSmodifications should lead to great reductions in thermal stability atthe terminus of the extended dsDNA replication intermediates (Jung et a.2016), and thus intrastrand hybridization (hairpin structure formation)can occur more readily, allowing more efficient exponentialamplification and the execution of LAMP at lower reaction temperatures.

To verify that exponential amplification during PS-LAMP is moreefficient than during regular LAMP, amplification performance wascompared at two different temperatures, 60° C. and 65° C.).Amplification for LAMP and PS-LAMP was carried out identically exceptfor the primers used, and was monitored in real-time using asequence-specific strand exchange reporter for 150 min in the absence orpresence of target template (1.2×10⁸ copies). FIG. 17a shows that at 60°C. LAMP could detect 1.2×10⁸ molecules within about 22 min, whilePS-LAMP required only 18 min to detect the same sample. At 65° C., thedetection times for LAMP and PS-LAMP were similar (22 and 24 min,respectively). Interestingly, the fluorescence intensity of the PS-LAMPreaction at 150 min was 1.8 times higher than that of LAMP at 60° C.(FIG. 17b ). This signal improvement at lower temperatures is consistentwith our hypothesis that phosphorothioates improve the self-folding ofloops.

(2) Optimization of lower temperature PS-LAMP reactions

Given these results, attempts to further optimize PS-LAMP for even lowertemperatures were made, which would also further its potential use as apoint-of-care diagnostic. When PS-LAMP was performed at progressivelylower temperatures (60, 55, 50 and 45° C.) no amplification waseventually observed at 45° C. (FIG. 24a ). The buffer and reactionconditions (4 mM of MgSO₄ and 8 U of Bst 2.0 DNA polymerase) weretherefore further optimized at 45° C. to see if a signal could begenerated. Magnesium ions are known to greatly impact self-folding(Zahran 2011; Sissi et al. 2009), and thus different concentrations ofmagnesium (0, 1, 2, 3 and 4 mM) were assessed. At 2 mM MgSO₄, a smallsignal increase was observed (FIG. 24b ). The amount of Bst 2.0 DNApolymerase was then increased from 8 U to 12 U and a thermostablesingle-stranded DNA binding protein (ET SSB) was added (0.5 μg) tofurther destabilize duplexes and promote self-folding at the termini(27-30). ET SSB proved more effective than an alternativesingle-stranded DNA binding protein, RecA (FIG. 23).

Using the optimized reaction conditions (FIG. 22), templateamplification by PS-LAMP was possible at 45° C., while normal LAMPreactions showed no amplification at this temperature (FIG. 17c ). Thatsaid, optimized PS-LAMP at 45° C. still took a longer time (63 min) tocome to completion than did a normal LAMP reaction at a highertemperatures (65° C., 22 min).

To further lower the operational temperature of PS-LAMP, urea was addedto the reaction mixture. Like heat and phosphorothioates, urea candisrupt base stacking and again improve the possibility of foldbackpriming (Singer et al. 2010; Conway et al. 1956; Schwinefus et al.2013). Upon optimization, a urea concentration of 1.44 M yielded aworkable PS-LAMP reaction at 40° C. (FIGS. 18a and 18b ). Both urea andlow temperatures decrease Bst 2.0 DNA polymerase activity, and thus theamount of Bst 2.0 DNA polymerase in the reaction was optimized onceagain, as shown in FIGS. 318c and 18d , and subsequent reactionscontained 60 U of polymerase; higher concentrations (80 U and 120 U)inhibited the reaction. At optimal urea and polymerase concentrations,ET SSB and MgSO₄ were re-optimized (0.5 μg and 2 mM, respectively (FIG.19), further decreasing the detection time from approximately 117 to 70min.

(3) Selectivity

To validate the sequence-specificity of PS-LAMP, various non-targettemplates (MERS 1a, NRP2 and human genomic DNA) were tested in parallelwith the target template (MERS 1b). As indicated in FIG. 25, at templateconcentrations of 500 μg (1.2×10⁸ copies) PS-LAMP produced negligibleresponses with non-cognate templates. When the target amplicon (MERS 1b)was mixed with the non-complementary templates, no diminution inpositive signal was observed (FIG. 25).

(4) Quantitation by PS-LAMP

The quantitative behavior of PS-LAMP at 40° C. in the presence of ureaand under fully optimized conditions was analyzed by monitoring thechanges in the fluorescence intensity oft he OSD reporter as a functionof template concentration. As shown in FIG. 20, when plasmids bearingthe MERS 1b and MERS 1a genes were used as targets, as few as 4,800molecules and 12 molecules, respectively, could be successfully detectedwithin 110 min and 80 min by PS-LAMP. The detection of the MERS 1aamplicon was roughly comparable with regular LAMP at 65° C., butdetection of the MERS 1b amplicon with PS-LAMP at 40° C. was lesssensitive than for regular LAMP at 65° C. (24 molecules within 30 min)(FIG. 20). This is not surprising, as there can be wide variation in thedetection limits for different amplicons even with regular LAMP,depending upon the sequences of primers and templates, and as thePS-LAMP technique is further developed it should be possible to identifycomparable optimization rules for amplicon choice and primer design.

d) Conclusions

LAMP is an ultrasensitive nucleic acid amplification method that canoften detect small numbers DNA or RNA templates within roughly an hour.However, the requirement for high temperatures limits it applicability,and nucleic acid hybridization chemistry, along with the disruption ofhelical stability by phosphorothioates have been relied upon to developPS-LAMP, which shows comparable sensitivities even down to 40° C.Operating at lower temperatures can inherently reduce device complexityand power consumption when adapting molecular diagnostics to microscaleor portable devices.

In addition to being able to operate at more moderate temperatures,PS-LAMP can better enable the use of degenerate primer sets to capture awider range of phylogenetic variants into amplicons. Improvingamplification at lower temperatures also enables the use of AT-richprimers and probes, which have previously proven problematic for LAMP(Tomita 2008).

Overall, PS-LAMP can now allow mesothermal amplification and concomitantapplications in molecular diagnostics that should be on par with similartechniques, such as RPA. As the technique is further developed, thedevelopment of primer design rules that accommodate lower temperaturesand phosphorothioates can increase its overall applicability, and theability to ‘dial in’ amplicon acquisition via the number ofphosphorothioates, the amount of urea present, and the temperature ofthe reaction provides much greater experimental control over theamplification and detection of specific targets.

3. Example 3: Zika RNA and PS-LAMP

PS-LAMP is ideal for use with amplifying RNA from Zika virus. Below arethe nucleic acids used with the polymerase for amplifying Zika:

zkNS5:  (SEQ ID NO: 6) CTAGTAACGGCCGCCAGTGTGCTGGAATTCGGTAGATCCATTGTGGTCCCTTGCCGCCACCAAGATGAATTGATTGGCCGAGCCCGTGTATCACCAGGGGCAGGATGGAGCATTCGGGAGACTGCCTGTCTAGCAAAATCATATGCACAGATGTGGCAGCTTCTTTACTTCCACAGAAGAGACCTTCGACTGATGGCCAATGCTATTTGTTCGGCTGTGCCAGTTGACTGGGTACCAACCGGGAGAACCACCTGGTCAATCCACGGAAAGGGAGAATGGATGACTACTGAGGACATGCTCATGGTGTGGAATAGAGTGTGGATTGAGGAGGAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGC zk.B3.dalt:  (SEQ ID NO: 7)GTCATCCARTCTCCRTTRCC zk.F3.1d  (SEQ ID NO: 8): ATCCATTGTGGTYCCYTGYzk.LR.1d  (SEQ ID NO: 9) TGCTCCATCCYGCCCCYGGHGA zk.BIP.1da1tPS: (SEQ ID NO: 10): A*T*G*C*V*C*A*R*A*T*G*T*G*G*C*A*G*C*T*Y*C*T*TCC CHG TTG GNA CCC A zk.FIP.1dPS  (SEQ ID NO: 11)G*C*H*A*G*R*C*A*R*G*C*A*G*T*C*T*C*M*C*G*R*GATGA AYTGATTGGCCGRGC

FIGS. 26A-D show optimization of RTX polymerase. Denaturation wascarried out at 65° C. for 5 minutes, then samples were transferred onice for at least two minutes. The temperature of amplification was 42 Cfor 3 hours. FIGS. 26A and B compare the performance of non-warm startRTX and warm-start RTX in urea condition for zika NS5. FIGS. 26C and 26Dshow formamide optimization. The best concentration of formamide wasshown to be 8%. FIG. 27 shows the final experimental protocol andresults. Formamide was used at 8%.

D. Tables

TABLE 1: Primers used for PCR, sequencing, cloning, and RCA ofThermostable Polymerase design. All Primers written in 5′ to 3′orientation. Selection primers were used in hbRCA as indicated. Primersare numbered consecutively as SEQ ID NO: 12-21 for JNM101, JNM135,JNM141, JNM219-222, JNM245, JNM258, and JNM259. Primers are numberedconsecutively as SEQ ID NO: 22-41 for JNM264-JNM283. Primers arenumbered consecutively as SEQ ID NOS: 42-45 for JNM309, JNM310, JNM316,and JNM317.

JNM101 TTTTCGTGTGAATATCAAGATCGC JNM135TTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCGGGAACTGCCAGACATCAAATAAAACAAAAGGC JNM141CTGGCCTTTTGCTCACATGACCCGACACCATCGAATGGCC GACAGTCATTCATCTTTCTGCC JNM219CAGACCCTAATTTCACATCATATGAC JNM220 CTTTTGCTCACATGACCCGA JNM221GTTTAACTTTAAGAAGGAGTAGGATCC JNM222 GAGGACAGAATTTGAATGCAAGC JNM245GCGATACAGACCCTAATTTCACATCATATGACAC JNM258 GTTCCTGAGAAAGACGGTGAGCG JNM259CGGGTTTTTACGTAAATCAGGTG Used in 20 Used in 10 Used in 2 primer RCAprimer RCA primer RCA hbRCA Primers (alternating F and R) (10F, 10R)(5F, 5R) (1F, 1R) JNM264 AAGCTTGCATTCAAATTCTGTCCTCAAG xx xx xx JNM265GGCCTTTTGCTCACATGACCC xx JNM266 GGACTATAAAGATACCAGGCGTTTCC xx JNM267GAACGACCTACACCGAACTGAGATAC xx xx JNM268 GCAGCAGCCACTGGTAACAG xx xxJNM269 CGGATCAAGAGCTACCAACTCTTTTTC xx JNM270 CTCAGTGGAACGAAAACTCACGTTAAGxx JNM271 CCTTGAATTGATCATATGCGGATTAGAAAAACAAC xx xx JNM272CTTTAGCGACTTGATGCTCTTGATCTTC xx xx JNM273 GTACACGGCCTACAGAAAAACAGTATG xxJNM274 GTATGGTGCCTATCTAACATCTCAATG xx JNM275 CAGCGCATTAGAGCTGCTTAATGAGGxx xx JNM276 CTCCCCGTCGTGTAGATAACTACG xx xx JNM277CGGATAAAGTTGCAGGACCACTTC xx JNM278 CGTTTGGTATGGCTTCATTCAGCTC xx JNM279CAGTGCTGCCATAACCATGAGTG xx xx JNM280 CAATACGGGATAATACCGCGCCA xx xxJNM281 GCTGGTGAAAGTAAAAGATGCTGAAGATC xx JNM282CAGGGTTATTGTCTCATGAGCGGATAC xx JNM283 GAGTGTTCACCGACAAACAACAGATAAAAC xxxx xx JNM309 CGCTTGAGGACAGAATTTTGGCAGAGGCAATTATCATTTCGCATCGTACCAAGTACTTC JNM310 CGCTTGAGGACAGAATTTTGGCAGAGGCAATTATCATTCTTTCGCAGATAACCAATCTTC JNM316 TTTGTTTAACTTTAAGAAGGAGATATACATATGGCTAGCAGAGGATCGCATCACCATCACCATCACATCG AAGGGCGCGAAAGTCCCAGCAGCGAG JNM317TTTGTTTAACTTTAAGAAGGAGATATACATATGGC TAGCAGAGGATCGCATCACCATCACCATCACATCGAAGGGCGCCTTCTTCACGAGTTCGGAC

TABLE 2  Primers and template used for qLAMP reactions forthermostable polymerase design. F3 GCCACCCAGAAGACTGTG B3TGGCAGGTTTTTCTAGACGG FIP CGCCAGTAGAGGCAGGGATGAGGGAAACTGTGGCGTGAT BIPGGTCATCCCTGAGCTGAACGGTCAGGTCCACCACTGACAC LR TGTTCTGGAGAGCCCCGCGGCC GAPDHCTAGTAACGGCCGCCAGTGTGCTGGAATTCCCACAGTCCA (Template)TGCCATCACTGCCACCCAGAAGACTGTGGATGGCCCCTCCGGGAAACTGTGGCGTGATGGCCGCGGGGCTCTCCAGAACATCATCCCTGCCTCTACTGGCGCTGCCAAGGCTGTGGGCAAGGTCATCCCTGAGCTGAACGGGAAGCTCACTGGCATGGCCTTCCGTGTCCCCACTGCCAACGTGTCAGTGGTGGACCTGACCTGCCGTCTAGAAAAACCTGCCAAATATGATGACATCAAGAAGGTGGTGAAGCAGGCGTCGGAGGGCCCCCTCAAGGGCATCCTGGGCTACACTGAGCACCAGGTGGTCTCCTCTGACTTCAACAGCGACACCCACTCCTCCACCTTTGACGCTGGGGCTGGCATTGCCCTCAACGACCACTTTGTCAAGCTCATTTCCTGGAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGA GC Primers are numberedconsecutively as SEQ ID NO: 46-51 in the table below.

TABLE 3  Oligonucleotide Sequences for PS-LAMP. MERS 1b primers TemplateTAATACGACTCACTATAGGGCGTGAATCTTAATTTACCCGCAAATGTCCCATACTCTCGTGTTATTTCCAGGATGGGCTTTAAACTCGATGCAACAGTTCCTGGATATCCTAAGCTTTTCATTACTCGTGAAGAGGCTGTAAGGCAAGTTCGAAGCTGGATAGGCTTCGATGTTGAGGGTGCTCATGCTTCCCGTAATGCATGTGGCACCAATGTGCCTCTACAATTAGGATTTTCAACTGGTGTGAACTTTGTTGTTCAGCCAGTTGGTGTTGTAGACACTGAGTCCTGACGAGTGGGTTTAACG (SEQ ID NO: 52) F3ACAGTTCCTGGATATCCTAAG (SEQ ID NO: 53) B3CTCAGTGTCTACAACACCA (SEQ ID NO: 54) FIPAGCACCCTCAACATCGAAGCACTCGTGAAGAGGCTGTA (SEQ ID NO: 55) BIPTGCTTCCCGTAATGCATGTGGACTGGCTGAACAACAAAGT (SEQ ID NO: 56) LoopCTATCCAGCTTCGAACTTGCCT (SEQ ID NO: 57)/56FAM/CACACCAGTTGAAAATCCTAATTGTAGAGGCACATTGGT OSD-FG/3InvdT/ (SEQ ID NO: 58)CTCTACAATTAGGATTTTCAACTGGTGTG/3IABkFQ/ (SEQ ID NO: OSDQ 59)MERS 1a primers Template TAATACGACTCACTATAGGGCTTGTGACTATGGCCTTCGTTATGTTGTTGGTTAAACACAAACACACCTTTTTGACACTTTTCTTGTTGCCTGTGGCTATTTGTTTGACTTATGCAAACATAGTCTACGAGCCCACTACTCCCATTTCGTCAGCGCTGATTGCAGTTGCAAATTGGCTTGCCCCCACTAATGCTTATATGCGCACTACACATACTGATATTGGTGTCTACATTAGTATGTCACTTGTATTAGTCATTGTAGTGAAGAGATTGTACAACCCATCACTTTCTAACTTTGCGTTAGCATTGTGCAGTGGTGTAATGTGGTTGTACACTTATAGCATTGGAGAAGCCTGACGAGTG GGTTTAACG (SEQ ID NO: 60)F3 TTATGCAAACATAGTCTACGAG (SEQ ID NO: 61) B3CGCAAAGTTAGAAAGTGATGG (SEQ ID NO: 62) FIPAAGCATTAGTGGGGGCAAGCCCCACTACTCCCATTTCG (SEQ ID NO: 63) BIPATGCGCACTACACATACTGATATTTGTACAATCTCTTCACT ACAATGA (SEQ ID NO: 64) LoopGGTGTCTACATTAGTATGTCACTTGTATTAG (SEQ ID NO: 65) OSD F/56FAM/CGAAGCCAATTTGCAACTGCAATCAGCGCTGAG/3I nvdT/ (SEQ ID NO: 66) OSD-QATTGCAGTTGCAAATTGGCTTCG/3IABkFQ/ (SEQ ID NO: 67) TemplateCACTCATTGGCACAGTGGTAGTTAGAGGTGAAAAGTAGAGCTGTCAAGCCCAAGGGCTTAGCTTTAGGGCTCCTCCTGAGTTCGGCCCACAGTAGAAGCAAGATTTTAACTAGCCCCTTTTCCTCTTCACCCTCCCATGATGCGCAGTGTTCAGAAAGCTGGTAAGTCCTAGGGATTTCCAGAAGTAGCCTGCAGAAGAAGGTAAGTTTGAAAGCCACTCCAGGGGTCCTGATGCTGTCATGCTCAGTGAGCCATTTTACAGTTCTCCAAAGTCTAGCCCTGTTTCGGACCTGCACTTCACCTCTAAGTTATGTACAACTCAACC (SEQ ID NO: 68) *Underlining indicates phosphorothioate(PS) modifications.

E. Polymerase Sequences

Primers used for PCR, sequencing, cloning, and RCA. All Primers writtenin 5′ to 3′ orientation. Selection primers were used in hbRCA asindicated.

SEQ ID NO: 3: MESPSSEEEKPLAKMAFTLADRVTEEMLADKAALVVEVVEENYHDAPIVGIAVVNEHGRFFLRPETALADPQFVAWLGDETKKKSMFDSKRAAVALKWKGIELCGVSFDLLLAAYLLDPAQGVDDVAAAAKMKQYEAVRPDEAVYGKGAKRAVPDEPVLAEHLVRKAAAIWELERPFLDELRRNEQDRLLVELEQPLSSILAEMEFAGVKVDTKRLEQMGKELAEQLGTVEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPYHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTKKVHTIFNQALTQTGRLSSTEPNLQNIPIRLEEGRKIRQAFVPSESDWLIFAADYSQIELRVLAHIAEDDNLMEAFRRDLDIHTKTAMDIFQVS EDEVTPNMRRQAKAVNFGIVYGISDYGLAQNLNISRKEAAEFIERYFESFPGVKRYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERMAMNTPIQGSAADIIKKAMIDLNARLKEERLQAHLLLQVHDELILEAPKEEMERLCRLVPEVMEQAVTLRVPLKVDYHYGSTWY DAK Klentaq (SEQ ID NO: 4)MLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE Variant 5.9 (v5.9)  (SEQ ID NO: 1)MASRGSHHHHHHIEGRLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIEGLLKVVRPDTKKVHTRFNQTATATGRLSSSDPNLQNIPVRTPLGORIRRAFIAGEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKSAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKENucleic Acid Encoding Variant 5.9 (v5.9)  (SEQ ID NO: 2)atggcgagccgcggcagccatcatcatcatcatcatattgaaggccgcctgctgcatgaantggcctgctggaaagcccgaaagcgctggaagaagcgccgtggccgccgccggaaggcgcgtttgtgggctttgtgctgagccgcaaagaaccgatgtgggcggatctgctggcgctggcggcggcgcgcggcggccgcgtgcatcgcgcgccggaaccgtataaagcgctgcgcgatctgaaagaagcgcgcggcctgctggcgaaagatctgagcgtgctggcgctgcgcgaaggcctgggcctgccgccgggcgatgatccgatgctgctggcgtatctgctggatccgagcaacaccaccccggaaggcgtggcgcgccgctatggcggcgaatggaccgaagaagcgggcgaacgcgcggcgctgagcgaacgcctgtttgcgaacctgtggggccgcctggaaggcgaagaacgcctgctgtggctgtatcgcgaagtggaacgcccgctgagcgcggtgctggcgcatatggaagcgaccggcgtgcgcctggatgtggcgtatctgcgcgcgctgagcctggaagtggcggaagaaattgcgcgcctggaagcggaagtgtttcgcctggcgggccatccgtttaacctgaacagccgcgatcagctggaacgcgtgctgtttgatgaactgggcctgccggcgattggcaaaaccgaaaaaaccggcaaacgcagcaccagcgcggcggtgctggaagcgctgcgcgaagcgcatccgattgtggaaaaaattctgcagtatcgcgaactgaccaaactgaaaagcacctatattgaaggcctgctgaaagtggtgcgcccggataccaaaaaagtgcatacccgctttaaccagaccgcgaccgcgaccggccgcctgagcagcagcgatccgaacctgcagaacattccggtgcgcaccccgctgggccagcgcattcgccgcgcgtttattgcgggcgaaggctggctgctggtggcgctggattatagccagattgaactgcgcgtgctggcgcatctgagcggcgatgaaaacctgattcgcgtgtttcaggaaggccgcgatattcataccgaaaccgcgagctggatgtttggcgtgccgcgcgaagcggtggatccgctgatgcgccgcgcggcgaaaaccattaactttggcgtgctgtatggcatgagcgcgcatcgcctgagccaggaactggcgattccgtatgaagaagcgcaggcgtttattgaacgctattttcagagctttccgaaagtgcgcgcgtggattgaaaaaaccctggaagaaggccgccgccgcggctatgtggaaaccctgtttggccgccgccgctatgtgccggatctggaagcgcgcgtgaaaagcgtgcgcgaagcggcggaacgcatggcgtttaacatgccggtgcagggcaccgcggcggatctgatgaaaagcgcgatggtgaaactgtttccgcgcctggaagaaatgggcgcgcgcatgctgctgcaggtgcatgatgaactggtgctggaagcgccgaaagaacgcgcggaagcggtggcgcgcctggcgaaagaagtgatggaaggcgtgtatccgctggcggtgccgctggaagtggaagtgggcattggcgaagattggctgagcgcgaaagaa Variant 7.16 (v7.16)  (SEQ ID NO: 5)MESPSSEEEKPLAKMAFTLADRVTEEMLADKAALVVEVVEENYHDAPIVGIAVVNEHGRFFLRPETALADPQFVAWLGDETKKKSMFDSKRAAVALKWKGIELCGVSFDLLLAAYLLDPAQGVDDVAAAAKMKQYEAVRPDEAVYGKGAKRAVPDEPVLAEHLVRKAAAIWELERPFLDELRRNEQDRLLVELEQPLSSILAEMEFAGVKVDTKRLEQMGKELAGQLGTVEQRIYELAGQEFNINSPKRLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPYHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTKKVHTIFNQALTQTGRLSSTEPNLQNIPIRLEEGRKIRQAFVPSESDWLIFAADYSQIELRVLAHIAEDDNLMEAFRRDLDIHTKTAMDIFQVSEDEVTPSMRRQAKAVNFGIVYGISDYGLAQNLNISRKEAAEFIGRYFESFPGVKRYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERMAMNTPIQGSAADIIKKAMIDLNARLKEERLQAHLLLQVHDELILEAPKEEMERLCRLVPEVMEQAVTLRVPLKVDYHYGSTWY DAK

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1-44. (canceled)
 45. A method of amplifying a nucleic acid, the methodcomprising exposing a target nucleic acid to a buffer solutioncomprising at least four primers, wherein at least one of the fourprimers comprises a phosphorothioated nucleotide; and amplifying thetarget nucleic acid using an isothermal amplification reaction, whereinthe isothermal amplification reaction produces at least one loopproduct, wherein at least part of the single-stranded portion of theloop product represents the target nucleic acid.
 46. The method of claim45, wherein the loop product is exposed to a strand displacementreporter, wherein the strand displacement reporter comprisessingle-stranded and double-stranded nucleic acid, and further wherein aportion of the single-stranded nucleic acid of the strand displacementreporter is complementary to at least a portion of the single-strandednucleic acid of the loop product representing the target nucleic acid,and allowing the loop product and the strand displacement reporter tointeract, wherein interaction between the strand displacement reporterand the target nucleic acid portion of the loop product produces adetectable signal, wherein the signal indicates the presence of thetarget nucleic acid.
 47. The method of claim 45, wherein the isothermalamplification reaction is loop-mediated isothermal amplification (LAMP),strand displacement amplification (SDA), polymerase spiral reaction(PSR), or helicase dependent amplification (HDA).
 48. (canceled) 49.(canceled)
 50. (canceled)
 51. The method of claim 45, wherein the buffercomprises denaturant.
 52. The method of claim 45, wherein the denaturantcomprises urea.
 53. The method of claim 45, wherein the buffer comprisesa DNA polymerase.
 54. The method of claim 45, wherein the buffercomprises a reverse transcriptase.
 55. (canceled)
 56. (canceled) 57.(canceled)
 58. (canceled)
 59. The method of claim 45, wherein the buffercomprises MgSO₄.
 60. (canceled)
 61. (canceled)
 62. The method of claim45, wherein the buffer comprises a Single-Stranded Binding (SSB)protein.
 63. (canceled)
 64. (canceled)
 65. The method of claim 45,wherein the strand displacement reporter is one step toeholddisplacement (OSD) reporter.
 66. The method of claim 45, wherein thetarget nucleic acid is RNA or DNA.
 67. The method of claim 45, whereinfour primers are used with the isothermal amplification reaction. 68.The method of claim 45, wherein five primers are used with theisothermal amplification reaction.
 69. The method of claim 45, whereinsix primers are used with the isothermal amplification reaction.
 70. Themethod of claim 45, wherein the method comprises using at least oneforward inner primer (FIP), at least one backward inner primer (BIP), atleast one forward outer primer (FOP), and at least one backward outerprimer (BOP).
 71. The method of claim 70, wherein at least one FIPcomprises at least one phosphorothioated nucleotide.
 72. The method ofclaim 70, wherein at least one BIP comprises at least onephosphorothioated nucleotide.
 73. The method of claim 45, wherein two ormore nucleotides of at least one primer are phosphorthioated.
 74. Themethod of claim 45, wherein the one or more phosphorothioatednucleotides are at the 5′-end of the primer.
 75. The method of claim 45,wherein the one or more phosphorothioated nucleotides are at the 3′-endof the primer. 76-96. (canceled)
 97. The method of claim 45, wherein thetarget nucleic acid is DNA.
 98. The method of claim 45, wherein thetarget nucleic acid is RNA.
 99. The method of claim 45, wherein theisothermal amplification is Loop-Mediated Isothermal Amplification(LAMP).
 100. The method of claim 99, wherein LAMP is PhosphorothioatedLAMP (PS-LAMP).
 101. The method of claim 99, wherein LAMP is ReverseTranscriptase LAMP (RT-LAMP).